Targeted mutagenesis of tobacco berberine bridge enzyme-like nucleic acids

ABSTRACT

The present invention relates to modifications of berberine bridge enzyme-like nucleic acids and their use in modulation of nicotine biosynthesis in plants.

STATEMENT OF PRIORITY

This application is a continuation application of U.S. application Ser.No. 17/532,438, filed on Nov. 22, 2021, which is a continuationapplication of U.S. application Ser. No. 16/778,780, filed on Jan. 31,2020, which is a continuation application of U.S. application Ser. No.15/780,354, filed on May 31, 2018, which is a 35 U.S.C. § 371 nationalphase application of International Application Serial No.PCT/US2016/064758, filed Dec. 2, 2016, which claims the benefit, under35 U.S.C. § 119 (a) of U.S. Provisional Patent Application No.62/263,151, filed Dec. 4, 2015, the entire contents of each of which areincorporated by reference herein.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

A Sequence Listing in XML format, entitled 5051-894CT3_ST26.xml, 51,739bytes in size, generated on Jun. 22, 2023 and filed herewith, is herebyincorporated by reference into the specification for its disclosures.

FIELD OF THE INVENTION

The present invention relates to modifications of berberine bridgeenzyme-like nucleic acids and their use in modulation of nicotinicalkaloid biosynthesis in plants.

BACKGROUND OF THE INVENTION

The pyridine alkaloids of tobacco (Nicotiana tabacum L.) are among themost studied group of plant secondary compounds. Nicotine constitutesgreater than 90% of the total alkaloid pool in most tobacco genotypesand is primarily responsible for the pharmacological responseexperienced by users of tobacco products. In decreasing order ofrelative abundance, the remaining major alkaloids in tobacco includeanatabine, nornicotine, and anabasine. Alkaloid levels in tobacco areinfluenced by environmental conditions, interactions with plant pests,and plant genetics.

Although nicotine is the primary compound that gives the users oftobacco products the pharmacological effect they seek, there are severalcircumstances where it would be desirable to develop products usingtobacco plants that produce and accumulate very low levels of nicotine.For example, some studies have shown that the use of low-nicotinecigarettes as a component in smoking cessation strategies can helpsmokers who are trying to quit (Hatsukami et al., 2010a; Donny et al.,2014). Other reports have demonstrated that by lowering the nicotinelevels below a critical threshold in tobacco products, they can nolonger initiate or maintain an addiction response (Benowitz andHenningfield, 1994; Benowitz et al., 2007). Studies such as these mayultimately influence regulatory agencies, such as the U.S. Food and DrugAdministration, who have been given the authority to determine whatacceptable levels of various tobacco constituents (including nicotine)will be allowable in cigarettes and other tobacco products.

Tobacco alkaloid levels are also of interest because of their role inthe production of tobacco specific nitrosamines (TSNAs), a potent groupof recognized carcinogens (Hecht, 1998, 2003; Hecht and Hoffman, 1989).The most important TSNAs are N-nitrosonornicotine (NNN) and4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which are derivedthrough nitrosation reactions with nornicotine and an oxidativederivative of nicotine (such as pseudooxynicotine), respectively, duringthe curing, storage, and consumption of tobacco. Because tobaccoalkaloids serve as precursors toward TSNA formation, low alkaloidtobacco plants have also been shown to produce reduced amounts of TSNAswithin the cured leaf (Xie et al., 2004). Further, while modificationsin the curing environment have led to substantial TSNA reductions influe-cured tobacco varieties, this has not been the case in theair-cured burley tobacco types.

This invention addresses the need for compositions and methods thatmodulate the nicotine biosynthesis pathway in plants.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method of producing aNicotiana plant having reduced nicotinic alkaloid content, comprisingintroducing into the plant (a) a mutation in an endogenous BBLepolynucleotide having 97% identity to the nucleotide sequence of SEQ IDNO:3, (b) a mutation in an endogenous BBLd-1 polynucleotide having 97%identity to the nucleotide sequence of SEQ ID NO:1, and/or (c) amutation in an endogenous BBLd-2 polynucleotide having 97% identity tothe nucleotide sequence of SEQ ID NO:2, thereby producing a plant havingreduced nicotinic alkaloid content.

In a second aspect, a plant having reduced nicotinic alkaloid content isprovided, the plant comprising a mutation in (a) a BBLe polynucleotidehaving 97% identity to the nucleotide sequence of SEQ ID NO:3, (b) aBBLd-1 polynucleotide having 97% identity to the nucleotide sequence ofSEQ ID NO:1, and/or (c) a BBLd-2 polynucleotide having 97% identity tothe nucleotide sequence of SEQ ID NO:2.

The present invention further provides plants and plant parts thereofproduced by the methods of the invention as well as crops and productsproduced from said plants and parts thereof. The present inventionfurther provides vectors and expression cassettes for carrying out themethods of the invention.

These and other aspects of the invention are set forth in more detail inthe description of the invention below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-1C show genome editing events using custom-designed meganucleaseconstructs. Wild type sequences in the targeted region of BBLe (FIG.1A), BBLd-1 (FIG. 1B) and BBLd-2 (FIG. 1C) are shown above the variousmutation events that were identified at that locus. Nucleotides in boldand italics show an exemplary 22 bp target site of a designer nuclease.Dashes represent nucleotides that have been deleted, and nucleotidesshaded grey represent small insertions. Numbers in parentheses at theright of each T₀ plant indicate the size of the deletion and/orinsertion event(s) in that plant. In T₀ plants with an asterisk, theindicated mutation is predicted to be biallelic (i.e. homozygous). Forall other T₀ plants the mutation is predicted to be monoallelic(heterozygous). T₀ events are underlined whose net loss or gain ofnucleotides at the mutation site is not divisible by three (thereforeensuring that reading frame downstream of the mutation would not becontiguous with the wild type reading frame).

DETAILED DESCRIPTION OF THE INVENTION

The present invention now will be described hereinafter with referenceto the accompanying drawings and examples, in which embodiments of theinvention are shown. This description is not intended to be a detailedcatalog of all the different ways in which the invention may beimplemented, or all the features that may be added to the instantinvention. For example, features illustrated with respect to oneembodiment may be incorporated into other embodiments, and featuresillustrated with respect to a particular embodiment may be deleted fromthat embodiment. Thus, the invention contemplates that in someembodiments of the invention, any feature or combination of features setforth herein can be excluded or omitted. In addition, numerousvariations and additions to the various embodiments suggested hereinwill be apparent to those skilled in the art in light of the instantdisclosure, which do not depart from the instant invention. Hence, thefollowing descriptions are intended to illustrate some particularembodiments of the invention, and not to exhaustively specify allpermutations, combinations and variations thereof.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

All publications, patent applications, patents and other referencescited herein are incorporated by reference in their entireties for theteachings relevant to the sentence and/or paragraph in which thereference is presented. References to techniques employed herein areintended to refer to the techniques as commonly understood in the art,including variations on those techniques or substitutions of equivalenttechniques that would be apparent to one of skill in the art.

Unless the context indicates otherwise, it is specifically intended thatthe various features of the invention described herein can be used inany combination. Moreover, the present invention also contemplates thatin some embodiments of the invention, any feature or combination offeatures set forth herein can be excluded or omitted. To illustrate, ifthe specification states that a composition comprises components A, Band C, it is specifically intended that any of A, B or C, or acombination thereof, can be omitted and disclaimed singularly or in anycombination.

As used in the description of the invention and the appended claims, thesingular forms “a,” “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.

As used herein, “and/or” refers to and encompasses any and all possiblecombinations of one or more of the associated listed items, as well asthe lack of combinations when interpreted in the alternative (“or”).

The term “about,” as used herein when referring to a measurable valuesuch as a dosage or time period and the like, refers to variations of±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

As used herein, phrases such as “between X and Y” and “between about Xand Y” should be interpreted to include X and Y. As used herein, phrasessuch as “between about X and Y” mean “between about X and about Y” andphrases such as “from about X to Y” mean “from about X to about Y.”

The terms “comprise,” “comprises” and “comprising” as used herein,specify the presence of the stated features, integers, steps,operations, elements, and/or components, but do not preclude thepresence or addition of one or more other features, integers, steps,operations, elements, components, and/or groups thereof.

As used herein, the transitional phrase “consisting essentially of”means that the scope of a claim is to be interpreted to encompass thespecified materials or steps recited in the claim and those that do notmaterially affect the basic and novel characteristic(s) of the claimedinvention. Thus, the term “consisting essentially of” when used in aclaim of this invention is not intended to be interpreted to beequivalent to “comprising.”

As used herein, “chimeric” refers to a nucleic acid molecule or apolypeptide in which at least two components are derived from differentsources (e.g., different organisms, different coding regions).

“Complement” as used herein can mean 100% complementarity or identitywith the comparator nucleotide sequence or it can mean less than 100%complementarity (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and the like, complementarity).

The terms “complementary” or “complementarity,” as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A.” Complementaritybetween two single-stranded molecules may be “partial,” in which onlysome of the nucleotides bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands.

As used herein, the terms “express,” “expresses,” “expressed” or“expression,” and the like, with respect to a nucleic acid moleculeand/or a nucleotide sequence (e.g., RNA or DNA) indicates that thenucleic acid molecule and/or a nucleotide sequence is transcribed and,optionally, translated. Thus, a nucleic acid molecule and/or anucleotide sequence may express, for example, a polypeptide of interestor a functional untranslated RNA.

A “fragment” or “portion” of a nucleotide sequence will be understood tomean a nucleotide sequence of reduced length relative (e.g., reduced by1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 ormore nucleotides) to a reference nucleic acid or nucleotide sequence andcomprising, consisting essentially of and/or consisting of a nucleotidesequence of contiguous nucleotides identical or substantially identical(e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% identical) to the reference nucleic acid or nucleotidesequence. Such a nucleic acid fragment or portion according to theinvention may be, where appropriate, included in a larger polynucleotideof which it is a constituent.

As used herein, the term “gene” refers to a nucleic acid moleculecapable of being used to produce mRNA, antisense RNA, RNAi (miRNA,siRNA, shRNA), anti-microRNA antisense oligodeoxyribonucleotide (AMO),and the like. Genes may or may not be capable of being used to produce afunctional protein or gene product. Genes can include both coding andnon-coding regions (e.g., introns, regulatory elements, promoters,enhancers, termination sequences and/or 5′ and 3′ untranslated regions).A gene may be “isolated” by which is meant a nucleic acid that issubstantially or essentially free from components normally found inassociation with the nucleic acid in its natural state. Such componentsinclude other cellular material, culture medium from recombinantproduction, and/or various chemicals used in chemically synthesizing thenucleic acid.

A “heterologous” or a “recombinant” nucleic acid is a nucleotidesequence not naturally associated with a host cell into which it isintroduced, including non-naturally occurring multiple copies of anaturally occurring nucleotide sequence. Alternatively, a heterologousnucleotide sequence can be one that does not naturally occur withanother nucleotide sequence to which it is associated. For example, anucleic acid construct comprising a “heterologous promoter” operablyassociated with a nucleic acid molecule is a promoter that does notnaturally occur with said nucleic acid molecule to which it isassociated.

Different nucleic acids or proteins having homology are referred toherein as “homologues.” The term homologue includes homologous sequencesfrom the same and other species and orthologous sequences from the sameand other species. “Homology” refers to the level of similarity betweentwo or more nucleic acid and/or amino acid sequences in terms of percentof positional identity (i.e., sequence similarity or identity). Homologyalso refers to the concept of similar functional properties amongdifferent nucleic acids or proteins. Thus, the compositions and methodsof the invention further comprise homologues to the nucleotide sequencesand polypeptide sequences of this invention. “Orthologous,” as usedherein, refers to homologous nucleotide sequences and/or amino acidsequences in different species that arose from a common ancestral geneduring speciation. A homologue of a nucleotide sequence of thisinvention has a substantial sequence identity (e.g., at least about 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, and/or 100%) to said nucleotide sequence of the invention. In someembodiments, an endogenous BBLe polynucleotide can have about 97%identity (e.g., about 97, 98, 99, or 100% identity) to the nucleotidesequence of SEQ ID NO:3, In some embodiments, an endogenous BBLd-1polynucleotide can have about 97% identity (e.g., about 97, 98, 99, or100% identity) to the nucleotide sequence of SEQ ID NO:1. In someembodiments, an endogenous BBLd-2 polynucleotide can have about 97%identity (e.g., about 97, 98, 99, or 100% identity) to the nucleotidesequence of SEQ ID NO:2.

As used herein, hybridization, hybridize, hybridizing, and grammaticalvariations thereof, refer to the binding of two fully complementarynucleotide sequences or substantially complementary sequences in whichsome mismatched base pairs may be present. The conditions forhybridization are well known in the art and vary based on the length ofthe nucleotide sequences and the degree of complementarity between thenucleotide sequences. In some embodiments, the conditions ofhybridization can be high stringency, or they can be medium stringencyor low stringency depending on the amount of complementarity and thelength of the sequences to be hybridized. The conditions that constitutelow, medium and high stringency for purposes of hybridization betweennucleotide sequences are well known in the art (See, e.g., Gasiunas etal. (2012) Proc. Natl. Acad. Sci. 109:E2579-E2586; M. R. Green and J.Sambrook (2012) Molecular Cloning: A Laboratory Manual. 4th Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, NY).

As used herein, the terms “increase,” “increasing,” “increased,”“enhance,” “enhanced,” “enhancing,” and “enhancement” (and grammaticalvariations thereof) describe an elevation of at least about 15%, 25%,50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more as compared to acontrol.

As used herein, “modify,” “modifying” or “modification” (and grammaticalvariations thereof) of a means any alteration of a BBL polynucleotide(e.g., BBLe, BBLd-1, BBLd-2) and/or BBL polypeptide or other polypeptideor polynucleotide that results in the reduction or elimination of theexpression of the nucleic acids and/or the production and/or activity ofthe polypeptides. Such modifications can include, but are not limitedto, deleting or inserting one or more nucleotides or an entire nucleicacid region (transcribed and untranscribed regions), and/or introducingone or more point mutations, which reduce or eliminate the expression ofthe nucleic acids and/or the production and/or activity of thepolypeptides.

As used herein, the terms “modulate,” “modulates,” “modulated” or“modulation” refer to enhancement (e.g., an increase) or inhibition(e.g., a reduction) in a specified activity (e.g., modulated nicotineproduction/content). Thus, in some embodiments, an elevation or increasein activity (e.g., nuclease activity) of about 15%, 25%, 50%, 75%, 100%,150%, 200%, 300%, 400%, 500% or more as compared to a control may beobserved. In other embodiments, a reduction in expression level oractivity (e.g., BBLe, BBLd-1, BBLd-2 expression level or BBLe, BBLd-1,BBLd-2 polypeptide activity) of about 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% ascompared to a control.

A “native” or “wild type” nucleic acid, nucleotide sequence, polypeptideor amino acid sequence refers to a naturally occurring or endogenousnucleic acid, nucleotide sequence, polypeptide or amino acid sequence.Thus, for example, a “wild type mRNA” is a mRNA that is naturallyoccurring in or endogenous to the organism. A “homologous” nucleic acidsequence is a nucleotide sequence naturally associated with a host cellinto which it is introduced.

Also as used herein, the terms “nucleic acid,” “nucleic acid molecule,”“nucleic acid construct,” “nucleotide sequence” and “polynucleotide”refer to RNA or DNA that is linear or branched, single or doublestranded, or a hybrid thereof. The term also encompasses RNA/DNAhybrids. When dsRNA is produced synthetically, less common bases, suchas inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and otherscan also be used for antisense, dsRNA, and ribozyme pairing. Forexample, polynucleotides that contain C-5 propyne analogues of uridineand cytidine have been shown to bind RNA with high affinity and to bepotent antisense inhibitors of gene expression. Other modifications,such as modification to the phosphodiester backbone, or the 2′-hydroxyin the ribose sugar group of the RNA can also be made. The nucleic acidconstructs of the present disclosure can be DNA or RNA, but arepreferably DNA. Thus, although the nucleic acid constructs of thisinvention may be described and used in the form of DNA, depending on theintended use, they may also be described and used in the form of RNA.

As used herein, “nicotinic alkaloid” refers to alkaloids derived fromnicotinic acid. These alkaloids generally contain a 3-pyridyl ringstructure, with nicotine, nornicotine, anatabine and anabasinerepresenting the predominant nicotinic alkaloids within the genusNicotiana. In some embodiments, a nicotinic alkaloid may comprise,consist essentially of, or consist of nicotine, nornicotine, anatabineand/or anabasine.

As used herein, “alkaloid content” means the total amount of alkaloidsfound in a plant, for example, in terms of percent dry weight (% dryweight) or percent fresh weight (% fresh weight).

A plant useful with this invention can be any Nicotiana plant thatproduces nicotine and/or other related alkaloids. Thus, in someembodiments, the plant can be Nicotiana tabacum, Nicotiana rustica orNicotiana benthamiana. Any variety of tobacco is useful with thisinvention including, but not limited to, Aromatic Fire-cured, Brightleaftobacco, Burley; Cavendish; Corojo; Criollo; Oriental Tobacco; Perique;Shade tobacco; Thuoc lao; Type 22; NC95, K326, K346, White Burley, WildTobacco, Y1, and the like.

As used herein, the term “nucleotide sequence” refers to a heteropolymerof nucleotides or the sequence of these nucleotides from the 5′ to 3′end of a nucleic acid molecule and includes DNA or RNA molecules,including cDNA, a DNA fragment or portion, genomic DNA, synthetic (e.g.,chemically synthesized) DNA, plasmid DNA, mRNA, and anti-sense RNA, anyof which can be single stranded or double stranded. The terms“nucleotide sequence” “nucleic acid,” “nucleic acid molecule,”“oligonucleotide” and “polynucleotide” are also used interchangeablyherein to refer to a heteropolymer of nucleotides. All nucleic acidsprovided herein have 5′ and 3′ ends. Further, except as otherwiseindicated, nucleic acid molecules and/or nucleotide sequences providedherein are presented herein in the 5′ to 3′ direction, from left toright and are represented using the standard code for representing thenucleotide characters as set forth in the U.S. sequence rules, 37 CFR §§1.821-1.825 and the World Intellectual Property Organization (WIPO)Standard ST.25.

As used herein, the term “percent sequence identity” or “percentidentity” refers to the percentage of identical nucleotides in a linearpolynucleotide sequence of a reference (“query”) polynucleotide molecule(or its complementary strand) as compared to a test (“subject”)polynucleotide molecule (or its complementary strand) when the twosequences are optimally aligned. In some embodiments, “percent identity”can refer to the percentage of identical amino acids in an amino acidsequence.

The term “plant part,” as used herein, includes but is not limited toreproductive tissues (e.g., petals, sepals, stamens, pistils,receptacles, anthers, pollen, flowers, fruits, flower bud, ovules,seeds, embryos); vegetative tissues (e.g., petioles, stems, roots, roothairs, root tips, pith, coleoptiles, stalks, shoots, branches, apicalmeristem, axillary bud, cotyledon, hypocotyls, and leaves); vasculartissues (e.g., phloem and xylem); specialized cells such as epidermalcells, parenchyma cells, chollenchyma cells, schlerenchyma cells,stomates, guard cells, cuticle, mesophyll cells; callus tissue; andcuttings. The term “plant part” also includes plant cells, includingplant cells that are intact in plants and/or parts of plants, plantprotoplasts, plant tissues, plant organs, plant cell tissue cultures,plant calli, plant clumps, and the like. As used herein, “shoot” refersto the above ground parts including the leaves and stems. As usedherein, the term “tissue culture” encompasses cultures of tissue, cells,protoplasts and callus.

As used herein, “plant cell” refers to a structural and physiologicalunit of the plant, which typically comprise a cell wall but alsoincludes protoplasts. A plant cell of the present invention can be inthe form of an isolated single cell or can be a cultured cell or can bea part of a higher-organized unit such as, for example, a plant tissue(including callus) or a plant organ. In some embodiments, a plant cellcan be an algal cell.

“Plant cell culture” means cultures of plant units such as, for example,protoplasts, cell culture cells, cells in plant tissues, pollen, pollentubes, ovules, embryo sacs, zygotes and embryos at various stages ofdevelopment. In some embodiments of the invention, a transgenic tissueculture or transgenic plant cell culture is provided, wherein thetransgenic tissue or cell culture comprises a nucleic acidmolecule/nucleotide sequence of the invention.

As used herein, a “plant organ” is a distinct and visibly structured anddifferentiated part of a plant such as a root, stem, leaf, flower bud,or embryo.

“Plant tissue” as used herein means a group of plant cells organizedinto a structural and functional unit. Any tissue of a plant in plantaor in culture is included. This term includes, but is not limited to,whole plants, plant organs, plant seeds, tissue culture and any groupsof plant cells organized into structural and/or functional units. Theuse of this term in conjunction with, or in the absence of, any specifictype of plant tissue as listed above or otherwise embraced by thisdefinition is not intended to be exclusive of any other type of planttissue.

As used herein, the terms “reduce,” “reduced,” “reducing,” “reduction,”“diminish,” “suppress,” and “decrease” (and grammatical variationsthereof), describe, for example, a decrease of at least about 5%, 10%,15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%as compared to a control. In particular embodiments, the reduction canresult in no or essentially no (i.e., an insignificant amount, e.g.,less than about 10% or even 5%) detectable activity or amount. Thus, forexample, reduced transcription of a target DNA can mean a reduction inthe transcription of the target gene of at least about 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% as compared to a control (e.g., a plant not comprising themutation in the BBL nucleic acids).

As used herein “sequence identity” refers to the extent to which twooptimally aligned polynucleotide or peptide sequences are invariantthroughout a window of alignment of components, e.g., nucleotides oramino acids. “Identity” can be readily calculated by known methodsincluding, but not limited to, those described in: ComputationalMolecular Biology (Lesk, A. M., ed.) Oxford University Press, New York(1988); Biocomputing: Informatics and Genome Projects (Smith, D. W.,ed.) Academic Press, New York (1993); Computer Analysis of SequenceData, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press,New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje,G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov,M. and Devereux, J., eds.) Stockton Press, New York (1991).

As used herein, a “target DNA,” “target region” or a “target region inthe genome” refers to a region of an organism's genome that is fullycomplementary or substantially complementary (e.g., at least 70%complementary (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more)) to a region of a gene againstwhich any class of custom designed nuclease (e.g., ZFN, TALEN,meganuclease, CRISPR-Cas and the like) has been engineered to bind andcleave.

As used herein, the phrase “substantially identical,” or “substantialidentity” in the context of at least two nucleic acid molecules,nucleotide sequences or protein sequences, refers to two or moresequences or subsequences that have at least about 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100%nucleotide or amino acid residue identity, when compared and aligned formaximum correspondence, as measured using one of the following sequencecomparison algorithms or by visual inspection.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for aligning a comparison window are wellknown to those skilled in the art and may be conducted by tools such asthe local homology algorithm of Smith and Waterman, the homologyalignment algorithm of Needleman and Wunsch, the search for similaritymethod of Pearson and Lipman, and optionally by computerizedimplementations of these algorithms such as GAP, BESTFIT, FASTA, andTFASTA available as part of the GCG® Wisconsin Package® (Accelrys Inc.,San Diego, CA). An “identity fraction” for aligned segments of a testsequence and a reference sequence is the number of identical componentswhich are shared by the two aligned sequences divided by the totalnumber of components in the reference sequence segment, i.e., the entirereference sequence or a smaller defined part of the reference sequence.Percent sequence identity is represented as the identity fractionmultiplied by 100. The comparison of one or more polynucleotidesequences may be to a full-length polynucleotide sequence or a portionthereof, or to a longer polynucleotide sequence. For purposes of thisinvention “percent identity” may also be determined using BLASTX version2.0 for translated nucleotide sequences and BLASTN version 2.0 forpolynucleotide sequences.

Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul et al., 1990). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when the cumulative alignment score falls off bythe quantity X from its maximum achieved value, the cumulative scoregoes to zero or below due to the accumulation of one or morenegative-scoring residue alignments, or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a wordlength (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci.USA 89: 10915 (1989)).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90: 5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a test nucleicacid sequence is considered similar to a reference sequence if thesmallest sum probability in a comparison of the test nucleotide sequenceto the reference nucleotide sequence is less than about 0.1 to less thanabout 0.001. Thus, in some embodiments of the invention, the smallestsum probability in a comparison of the test nucleotide sequence to thereference nucleotide sequence is less than about 0.001.

Two nucleotide sequences can also be considered to be substantiallycomplementary when the two sequences hybridize to each other understringent conditions. In some representative embodiments, two nucleotidesequences considered to be substantially complementary hybridize to eachother under highly stringent conditions.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern hybridizations are sequence dependent, andare different under different environmental parameters. An extensiveguide to the hybridization of nucleic acids is found in TijssenLaboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes part I chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays” Elsevier, New York (1993). Generally, highlystringent hybridization and wash conditions are selected to be about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH.

The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe. Very stringent conditions are selected to be equal to the T_(m)for a particular probe. An example of stringent hybridization conditionsfor hybridization of complementary nucleotide sequences which have morethan 100 complementary residues on a filter in a Southern or northernblot is 50% formamide with 1 mg of heparin at 42° C., with thehybridization being carried out overnight. An example of highlystringent wash conditions is 0.1 5M NaCl at 72° C. for about 15 minutes.An example of stringent wash conditions is a 0.2×SSC wash at 65° C. for15 minutes (see, Sambrook, infra, for a description of SSC buffer).Often, a high stringency wash is preceded by a low stringency wash toremove background probe signal. An example of a medium stringency washfor a duplex of, e.g., more than 100 nucleotides, is 1×SSC at 45° C. for15 minutes. An example of a low stringency wash for a duplex of, e.g.,more than 100 nucleotides, is 4-6×SSC at 40° C. for 15 minutes. Forshort probes (e.g., about 10 to 50 nucleotides), stringent conditionstypically involve salt concentrations of less than about 1.0 M Na ion,typically about 0.01 to 1.0 M Na ion concentration (or other salts) atpH 7.0 to 8.3, and the temperature is typically at least about 30° C.Stringent conditions can also be achieved with the addition ofdestabilizing agents such as formamide. In general, a signal to noiseratio of 2× (or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates detection of a specifichybridization. Nucleotide sequences that do not hybridize to each otherunder stringent conditions are still substantially identical if theproteins that they encode are substantially identical. This can occur,for example, when a copy of a nucleotide sequence is created using themaximum codon degeneracy permitted by the genetic code.

The following are examples of sets of hybridization/wash conditions thatmay be used to clone homologous nucleotide sequences that aresubstantially identical to reference nucleotide sequences of theinvention. In one embodiment, a reference nucleotide sequence hybridizesto the “test” nucleotide sequence in 7% sodium dodecyl sulfate (SDS),0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50°C. In another embodiment, the reference nucleotide sequence hybridizesto the “test” nucleotide sequence in 7% sodium dodecyl sulfate (SDS),0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 50°C. or in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50°C. with washing in 0.5×SSC, 0.1% SDS at 50° C. In still furtherembodiments, the reference nucleotide sequence hybridizes to the “test”nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 50° C., or in 7%sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. withwashing in 0.1×SSC, 0.1% SDS at 65° C.

In any of the embodiments described herein, the nucleotide sequences ofinterest (e.g., nucleic acids encoding nucleases useful for mutating BBLnucleic acids) can be operably associated with a variety of promoters,terminators, and/or other regulatory elements for expression in plantcell. Any promoter, terminator or other regulatory element functional ina plant cell may be used with the nucleic acids of this invention. Inrepresentative embodiments, a promoter may be operably linked to apolynucleotide and/or nucleic acid useful in carrying out the invention.In some embodiments, a terminator may be operably linked to apolynucleotide and/or nucleic acid of the invention.

A “promoter” is a nucleotide sequence that controls or regulates thetranscription of a nucleotide sequence (i.e., a coding sequence) that isoperably associated with the promoter. The coding sequence may encode apolypeptide and/or a functional RNA. Typically, a “promoter” refers to anucleotide sequence that contains a binding site for RNA polymerase IIor RNA polymerase III and directs the initiation of transcription. Ingeneral, promoters are found 5′, or upstream, relative to the start ofthe coding region of the corresponding coding sequence. The promoterregion may comprise other elements that act as regulators of geneexpression. These include a TATA box consensus sequence, and often aCAAT box consensus sequence (Breathnach and Chambon, (1981) Annu. Rev.Biochem. 50:349). In plants, the CAAT box may be substituted by the AGGAbox (Messing et al., (1983) in Genetic Engineering of Plants, T. Kosuge,C. Meredith and A. Hollaender (eds.), Plenum Press, pp. 211-227).

Promoters useful with this invention can include, for example,constitutive, inducible, temporally regulated, developmentallyregulated, chemically regulated, tissue-preferred and/or tissue-specificpromoters for use in the preparation of recombinant nucleic acidmolecules, i.e., “chimeric genes” or “chimeric polynucleotides.” Thesevarious types of promoters are known in the art. The choice of promoterwill vary depending on the temporal and spatial requirements forexpression, and also depending on the host cell to be transformed.Promoters for many different organisms are well known in the art. Basedon the extensive knowledge present in the art, the appropriate promotercan be selected for the particular host organism of interest. Thus, forexample, much is known about promoters upstream of highly constitutivelyexpressed genes in model organisms and such knowledge can be readilyaccessed and implemented in other systems as appropriate. In someembodiments, expression of a nucleotide sequence of interest can be inany plant and/or plant part, (e.g., in leaves, in stalks or stems, inears, in inflorescences, in roots, seeds and/or seedlings, and the like)and the promoter is selected accordingly.

In embodiments described herein, one or more of the polynucleotides andnucleic acids of the invention may be operably associated with apromoter as well as a terminator, and/or other regulatory elements forexpression in plant cell. Any promoter, terminator or other regulatoryelement that is functional in a plant cell may be used with the nucleicacids of this invention. Non-limiting examples of promoters useful withthis invention include an Arabidopsis thaliana U6 RNA polymerase IIIpromoter, a 35S promoter, actin promoter, ubiquitin promoter, Rubiscosmall subunit promoter, an inducible promoter, including but not limitedto, a an AlcR/AlcA (ethanol inducible) promoter, a glucocorticoidreceptor (GR) fusion, GVG, a pOp/LhGR (dexamethasone inducible)promoter, a XVE/OlexA (β-estradiol inducible) promoter, a heat shockpromoter and/or a bidirectional promoter (See, e.g., Gatz, Christine.Current Opinion in Biotechnology 7(2):168-172 (1996); Borghi L. MethodsMol Biol. 655:65-75(2010); Baron et al. Nucleic acids research 23(17)995), 3605; Kumar et al. Plant molecular biology 87(4-5):341-353(2015)).

By “operably linked” or “operably associated” as used herein, it ismeant that the indicated elements are functionally related to eachother, and are also generally physically related. Thus, the term“operably linked” or “operably associated” as used herein, refers tonucleotide sequences on a single nucleic acid molecule that arefunctionally associated. Thus, a first nucleotide sequence that isoperably linked to a second nucleotide sequence means the firstnucleotide sequence is placed in a functional relationship with thesecond nucleotide sequence. For instance, a promoter is operablyassociated with a nucleotide sequence if the promoter effects thetranscription or expression of said nucleotide sequence. Those skilledin the art will appreciate that the control sequences (e.g., promoter)need not be contiguous with the nucleotide sequence to which it isoperably associated, as long as the control sequences function to directthe expression thereof. Thus, for example, intervening untranslated, yettranscribed, sequences can be present between a promoter and anucleotide sequence, and the promoter can still be considered “operablylinked” to the nucleotide sequence.

In some embodiments, the components for modifying or mutating a BBLnucleic acid and any other polynucleotide of interest (e.g., otherpolynucleotides encoding nicotinic alkaloid biosynthetic enzymestranscription factors that positively regulate nicotinic alkaloidbiosynthesis) may be comprised in an “expression cassette.” As usedherein, “expression cassette” means a nucleic acid construct comprisinga nucleotide sequence of interest (e.g., a nuclease useful for mutatinga BBL nucleic acid), wherein said nucleotide sequence is operativelyassociated with at least a control sequence (e.g., a promoter). Anexpression cassette may be chimeric, meaning that at least one of itscomponents is heterologous with respect to at least one of its othercomponents. Thus, for example, the nucleic acids to be expressed may beoperably linked to a promoter or other regulatory element that isheterologous to the nucleic acids to be expressed (e.g., heterologous toa CRISPR guide DNA). An expression cassette may also be one that isnaturally occurring but has been obtained in a recombinant form usefulfor heterologous expression.

In addition to promoters, an expression cassette also can optionallyinclude additional regulatory elements functional in a plant cellincluding, but not limited to, a transcriptional and/or translationaltermination region (i.e., termination region). A variety oftranscriptional terminators are available for use in expressioncassettes and are responsible for the termination of transcriptionbeyond the heterologous nucleotide sequence of interest and correct mRNApolyadenylation. The termination region may be native to thetranscriptional initiation region, may be native to the operably linkednucleotide sequence of interest, may be native to the host cell, or maybe derived from another source (i.e., foreign or heterologous to thepromoter, to the nucleotide sequence of interest, to the host, or anycombination thereof). Non-limiting examples of terminators functional ina plant and useful with this invention include an actin terminator; aRubisco small subunit terminator, a Rubisco large subunit terminator, anopaline synthase terminator, and/or a ubiquitin terminator.

A number of non-translated leader sequences derived from viruses areknown to enhance gene expression. Specifically, leader sequences fromTobacco Mosaic Virus (TMV, the “ω-sequence”), Maize Chlorotic MottleVirus (MCMV) and Alfalfa Mosaic Virus (AMV) have been shown to beeffective in enhancing expression (Gallie et al. (1987) Nucleic AcidsRes. 15:8693-8711; and Skuzeski et al. (1990) Plant Mol. Biol.15:65-79). Other leader sequences known in the art include, but are notlimited to, picornavirus leaders such as an encephalomyocarditis (EMCV)5′ noncoding region leader (Elroy-Stein et al. (1989) Proc. Natl. Acad.Sci. USA 86:6126-6130); potyvirus leaders such as a Tobacco Etch Virus(TEV) leader (Allison et al. (1986) Virology 154:9-20); Maize DwarfMosaic Virus (MDMV) leader (Allison et al. (1986), supra); humanimmunoglobulin heavy-chain binding protein (BiP) leader (Macejak & Samow(1991) Nature 353:90-94); untranslated leader from the coat protein mRNAof AMV (AMV RNA 4; Jobling & Gehrke (1987) Nature 325:622-625); tobaccomosaic TMV leader (Gallie et al. (1989) Molecular Biology of RNA237-256); and MCMV leader (Lommel et al. (1991) Virology 81:382-385).See also, Della-Cioppa et al. (1987) Plant Physiol. 84:965-968.

An expression cassette also can optionally include a transcriptionaland/or translational termination region (i.e., termination region) thatis functional in plants. A variety of transcriptional terminators areavailable for use in expression cassettes and are responsible for thetermination of transcription beyond the heterologous nucleotide sequenceof interest and correct mRNA polyadenylation. The termination region maybe native to the transcriptional initiation region, may be native to theoperably linked nucleotide sequence of interest, may be native to theplant host, or may be derived from another source (i.e., foreign orheterologous to the promoter, the nucleotide sequence of interest, theplant host, or any combination thereof). Appropriate transcriptionalterminators include, but are not limited to, the CAMV 35S terminator,the tml terminator, the nopaline synthase terminator and/or the pea rbcsE9 terminator. These can be used in both monocotyledons anddicotyledons. In addition, a coding sequence's native transcriptionterminator can be used.

An expression cassette also can include a nucleotide sequence for aselectable marker, which can be used to select a transformed host cell.As used herein, “selectable marker” means a nucleotide sequence thatwhen expressed imparts a distinct phenotype to the host cell expressingthe marker and thus allows such transformed cells to be distinguishedfrom those that do not have the marker. Such a nucleotide sequence mayencode either a selectable or screenable marker, depending on whetherthe marker confers a trait that can be selected for by chemical means,such as by using a selective agent (e.g., an antibiotic and the like),or on whether the marker is simply a trait that one can identify throughobservation or testing, such as by screening (e.g., fluorescence). Manyexamples of suitable selectable markers are known in the art and can beused in the expression cassettes described herein.

In addition to expression cassettes, the nucleic acids described hereincan be used in connection with vectors. The term “vector” refers to acomposition for transferring, delivering or introducing one or morenucleic acids into a cell. A vector comprises a nucleic acid moleculecomprising the nucleotide sequence(s) to be transferred, delivered orintroduced. Vectors for use in transformation of host organisms are wellknown in the art. Non-limiting examples of general classes of vectorsinclude but are not limited to a viral vector, a plasmid vector, a phagevector, a phagemid vector, a cosmid vector, a fosmid vector, abacteriophage, an artificial chromosome, or an Agrobacterium binaryvector in double or single stranded linear or circular form which may ormay not be self transmissible or mobilizable. A vector as defined hereincan transform a eukaryotic host either by integration into the cellulargenome or exist extrachromosomally (e.g. autonomous replicating plasmidwith an origin of replication). Additionally included are shuttlevectors by which is meant a DNA vehicle capable, naturally or by design,of replication in two different host organisms. In some representativeembodiments, the nucleic acid in the vector is under the control of, andoperably linked to, an appropriate promoter or other regulatory elementsfor transcription in a host cell. The vector may be a bi-functionalexpression vector which functions in multiple hosts. In the case ofgenomic DNA, this may contain its own promoter or other regulatoryelements and in the case of cDNA this may be under the control of anappropriate promoter or other regulatory elements for expression in thehost cell. Accordingly, the polynucleotides and/or expression cassettescan be comprised in vectors as described herein and as known in the art.

“Introducing,” “introduce,” “introduced” (and grammatical variationsthereof) in the context of a polynucleotide of interest (e.g., anuclease useful for mutating a BBL nucleic acid) means presenting thepolynucleotide of interest to the host organism or cell of said organism(e.g., host cell) in such a manner that the polynucleotide gains accessto the interior of a cell. Where more than one polynucleotide is to beintroduced these polynucleotides can be assembled as part of a singlepolynucleotide or nucleic acid construct, or as separate polynucleotidesor nucleic acid constructs, and can be located on the same or differentexpression constructs or transformation vectors. Accordingly, thesepolynucleotides can be introduced into cells in a single transformationevent, in separate transformation/transfection events, or, for example,they can be incorporated into an organism by conventional breedingprotocols. Thus, in some aspects, one or more polynucleotides encodingnucleases useful for modifying or mutating a BBL nucleic acid (e.g.,Crispr-Cas nucleases, meganucleases, zinc finger nucleases (ZFNs),and/or transcription activator-like effector nucleases (TALENs)) can beintroduced singly or in combination in a single expression cassetteand/or vector into a host organism or a cell of said host organism.

The term “transformation” or “transfection” as used herein refers to theintroduction of a heterologous nucleic acid into a cell, such as anucleic acid encoding a nuclease. Transformation of a cell may be stableor transient or may be in part stably transformed and in parttransiently transformed. Thus, in some embodiments, the modifications tothe plant genome can be stable and in some embodiments, themodifications can be transient. In some embodiments, after stabletransformation, the nucleic acid constructs introduced to the plantgenome can be removed by, for example, crossing with non-modified plantsor segregation of non-homozygous plants.

“Transient transformation” in the context of a polynucleotide means thata polynucleotide is introduced into the cell and does not integrate intothe genome of the cell.

By “stably introducing” or “stably introduced,” in the context of apolynucleotide, means that the introduced polynucleotide is stablyincorporated into the genome of the cell, and thus the cell is stablytransformed with the polynucleotide.

“Stable transformation” or “stably transformed” as used herein meansthat a nucleic acid construct is introduced into a cell and integratesinto the genome of the cell. As such, the integrated nucleic acidconstruct is capable of being inherited by the progeny thereof, moreparticularly, by the progeny of multiple successive generations.“Genome” as used herein can include the nuclear, plastid, and/ormitochondrial genome, and therefore may include integration of a nucleicacid construct into the nuclear, plastid and/or mitochondrial genome.Stable transformation as used herein may also refer to a transgene thatis maintained extrachromasomally, for example, as a minichromosome or aplasmid.

Transient transformation may be detected by, for example, anenzyme-linked immunosorbent assay (ELISA) or Western blot, which candetect the presence of a peptide or polypeptide encoded by one or moretransgene introduced into a plant or plant cell. Stable transformationof a cell can be detected by, for example, a Southern blot hybridizationassay of genomic DNA of the cell with nucleic acid sequences whichspecifically hybridize with a nucleotide sequence of a transgeneintroduced into an organism (e.g., a bacterium, an archaea, a yeast, analgae, and the like). Stable transformation of a cell can be detectedby, for example, a Southern blot hybridization assay of DNA of the cellwith nucleic acid sequences which specifically hybridize with anucleotide sequence of a transgene introduced into a plant or otherorganism. Stable transformation of a cell can also be detected by, e.g.,a polymerase chain reaction (PCR) or other amplification reactions asare well known in the art, employing specific primer sequences thathybridize with target sequence(s) of a transgene, resulting inamplification of the transgene sequence, which can be detected accordingto standard methods. Transformation can also be detected by directsequencing and/or hybridization protocols well known in the art.

Procedures for transforming plants are well known and routine in the artand are described throughout the literature. Non-limiting examples ofmethods for transformation of plants include transformation viabacterial-mediated nucleic acid delivery (e.g., via Agrobacteria),viral-mediated nucleic acid delivery, silicon carbide or nucleic acidwhisker-mediated nucleic acid delivery, liposome mediated nucleic aciddelivery, microinjection, microparticle bombardment,calcium-phosphate-mediated transformation, cyclodextrin-mediatedtransformation, electroporation, nanoparticle-mediated transformation,sonication, infiltration, PEG-mediated nucleic acid uptake, as well asany other electrical, chemical, physical (mechanical) and/or biologicalmechanism that results in the introduction of nucleic acid into theplant cell, including any combination thereof. General guides to variousplant transformation methods known in the art include Miki et al.(“Procedures for Introducing Foreign DNA into Plants” in Methods inPlant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J.E., Eds. (CRC Press, Inc., Boca Raton, 1993), pages 67-88) andRakowoczy-Trojanowska (Cell. Mol. Biol. Lett. 7:849-858 (2002)).

Agrobacterium-mediated transformation is a commonly used method fortransforming plants, in particular, dicot plants, because of its highefficiency of transformation and because of its broad utility with manydifferent species. Agrobacterium-mediated transformation typicallyinvolves transfer of the binary vector carrying the foreign DNA ofinterest to an appropriate Agrobacterium strain that may depend on thecomplement of vir genes carried by the host Agrobacterium strain eitheron a co-resident Ti plasmid or chromosomally (Uknes et al. (1993) PlantCell 5:159-169). The transfer of the recombinant binary vector toAgrobacterium can be accomplished by a triparental mating procedureusing Escherichia coli carrying the recombinant binary vector, a helperE. coli strain that carries a plasmid that is able to mobilize therecombinant binary vector to the target Agrobacterium strain.Alternatively, the recombinant binary vector can be transferred toAgrobacterium by nucleic acid transformation (Höfgen & Willmitzer (1988)Nucleic Acids Res. 16:9877).

Transformation of a plant by recombinant Agrobacterium usually involvesco-cultivation of the Agrobacterium with explants from the plant andfollows methods well known in the art. Transformed tissue is regeneratedon selection medium carrying an antibiotic or herbicide resistancemarker between the binary plasmid T-DNA borders.

Another method for transforming plants, plant parts and/or plant cellsinvolves propelling inert or biologically active particles at planttissues and cells. See, e.g., U.S. Pat. Nos. 4,945,050; 5,036,006 and5,100,792. Generally, this method involves propelling inert orbiologically active particles at the plant cells under conditionseffective to penetrate the outer surface of the cell and affordincorporation within the interior thereof. When inert particles areutilized, the vector can be introduced into the cell by coating theparticles with the vector containing the nucleic acid of interest.Alternatively, a cell or cells can be surrounded by the vector so thatthe vector is carried into the cell by the wake of the particle.Biologically active particles (e.g., dried yeast cells, dried bacteriumor a bacteriophage, each containing one or more nucleic acids sought tobe introduced) also can be propelled into plant tissue.

A nucleotide sequence therefore can be introduced into the plant, plantpart and/or plant cell in any number of ways that are well known in theart. The methods of the invention do not depend on a particular methodfor introducing one or more nucleotide sequences into a plant, only thatthey gain access to the interior of at least one cell of the plant.Thus, in particular embodiments of the invention, intact plants can beregenerated from these transformed cells using any of a variety of knowntechniques. Plant regeneration from plant cells, plant tissue cultureand/or cultured protoplasts is described, for example, in Evans et al.(Handbook of Plant Cell Cultures, Vol. 1, MacMilan Publishing Co. NewYork (1983)); and Vasil I. R. (ed.) (Cell Culture and Somatic CellGenetics of Plants, Acad. Press, Orlando, Vol. I (1984), and Vol. II(1986)). Methods of selecting for transformed transgenic plants, plantcells and/or plant tissue culture are routine in the art and can beemployed in the methods of the invention provided herein.

As used herein, “tobacco product” refers to a product comprisingmaterial produced by a Nicotiana plant, including for example, nicotinegum and patches for smoking cessation, cigarette tobacco includingexpanded (puffed) and reconstituted tobacco, cigar tobacco, pipetobacco, cigarettes, cigars, and all forms of smokeless tobacco such aschewing tobacco, snuff, snus and lozenges. “Cigarettes” includeselectronic cigarettes and “heat not burn” products which arecigarette-like devices that heat tobacco rather than burn tobacco.

The present invention is directed in part to the discovery thatmodifying polynucleotides encoding BBLe, BBLd-1 or BBLd-2 so as toreduce or eliminate expression and/or activity of the polynucleotidesand/or any polypeptide produced can result in the plant having reducednicotinic alkaloid content as compared to a plant that does not comprisesaid modification.

Since the 1930s, tobacco researchers have been engaged in alteringtobacco genetics to affect the levels of nicotine and other alkaloids.Large reductions in tobacco alkaloid levels have historically beenachieved through the use of naturally-occurring recessive alleles at theNic1 and Nic2 loci (also designated as the A and B loci, in someliterature). Recessive alleles at both of these loci can reduce alkaloidlevels from between 1.5% and 4.5% total dry weight to as low asapproximately 0.2% (Legg et al., 1969; Legg and Collins, 1971; Chaplinand Weeks, 1976). Although the Nic1 locus remains uncharacterized, theNic2 locus was recently shown to encode for a cluster of transcriptionfactors of the ethylene response factor (ERF) gene family (Shoji et al.,2010). However, tobacco varieties carrying the recessive nic1 or nic2alleles have not been widely used due to negative associations withyield and quality (Legg et al., 1970; Chaplin and Weeks, 1976; Chaplinand Burk, 1983). The negative attributes of tobacco plants homozygousfor the mutant nic1 and nic2 loci are likely a result of the fact thatthey not only influence the expression of genes involved in alkaloidbiosynthesis but an array of unrelated genes as well (Kidd et al.,2006).

A great deal of knowledge has been gained over the last twenty yearsregarding the molecular biology underlying the biosynthesis of tobaccoalkaloids (reviewed by Dewey and Xie, 2013). As the genes encodingspecific steps of nicotine biosynthesis have been elucidated, these havebecome targets for reducing the nicotine content of the plant. Althoughit has been shown that transgene mediated down-regulation of severalsteps in the alkaloid biosynthetic pathway can result in reducednicotine accumulation, most of these perturbations are accompanied byundesirable side-effects, such as reduced growth, or the concomitantincrease in the levels of the typically minor alkaloid anatabine(reviewed in Dewey and Xie, 2013). In tobacco, enzymes referred to asberberine bridge enzyme-like (BBL) proteins catalyze one of last stepsof alkaloid biosynthesis (Kajikawa et al., 2011). Our research to dateon this gene family, summarized below, has shown that this specific stepof the alkaloid biosynthetic pathway is a particularly attractive targetfor the development of high quality, low alkaloid tobaccos.

Thus, in some embodiments of the invention, a method of producing aNicotiana plant or plant part having reduced nicotinic alkaloid contentis provided, comprising, consisting essentially of, or consisting ofintroducing into a Nicotiana plant or plant part (a) a mutation in anendogenous BBLe polynucleotide having 97% identity to the nucleotidesequence of SEQ ID NO:3, (b) a mutation in an endogenous BBLd-1polynucleotide having 97% identity to the nucleotide sequence of SEQ IDNO:1, and/or (c) a mutation in an endogenous BBLd-2 polynucleotidehaving 97% identity to the nucleotide sequence of SEQ ID NO:2, therebyproducing a Nicotiana plant or plant part having reduced nicotinicalkaloid content as compared to a control Nicotiana plant or plant partthat does not comprise the mutation of (a), (b), or (c). In someembodiments, the at least one recombinant nucleic acid may be stablyincorporated into the genome of the plant, which is then removed fromthe genome of the plant by backcrossing the transgenic Nicotiana plantwith a Nicotiana plant not comprising the at least one recombinantnucleic acid. In some embodiments, introducing comprises introducing themutation into a plant cell and regenerating the plant cell into a plant,thereby producing a plant having reduced nicotinic alkaloid content. Inother embodiments, the method further comprises selecting a Nicotianaplant or plant part comprising (a) a mutation in the BBLe nucleotidesequence having at least 97% identity to the nucleotide sequence of SEQID NO:3, (b) a mutation in the BBLd-1 polynucleotide sequence having atleast 97% identity to the nucleotide sequence of SEQ ID NO:1 (BBLd-1),and/or (c) a mutation in the BBLd-2 polynucleotide sequence having atleast 97% identity to the nucleotide sequence of SEQ ID NO:2 (BBLd-2).In some embodiments, the nicotinic alkaloid comprises, consistsessentially of, or consists of nicotine.

In some embodiments, a method of reducing nicotinic alkaloid content ina Nicotiana plant or plant part is provided, comprising, consistingessentially of, or consisting of introducing into a Nicotiana plant orplant part (a) a mutation in an endogenous BBLe polynucleotide having97% identity to the nucleotide sequence of SEQ ID NO:3, (b) a mutationin an endogenous BBLd-1 polynucleotide having 97% identity to thenucleotide sequence of SEQ ID NO:1, and/or (c) a mutation in anendogenous BBLd-2 polynucleotide having 97% identity to the nucleotidesequence of SEQ ID NO:2, thereby producing a Nicotiana plant or plantpart having reduced nicotinic alkaloid content as compared to a controlNicotiana plant or plant part that does not comprise the mutation of(a), (b), or (c). In some embodiments, the at least one recombinantnucleic acid may be stably incorporated into the genome of the plant,which is then removed from the genome of the plant by backcrossing theplant with a plant not comprising the at least one recombinant nucleicacid. In further embodiments, introducing comprises introducing themutation into a plant cell and regenerating the plant cell into a plantor plant part, thereby producing a plant or plant part having reducednicotinic alkaloid content. In other embodiments, the method furthercomprises selecting a Nicotiana plant or plant part comprising (a) amutation in the BBLe nucleotide sequence having at least 97% identity tothe nucleotide sequence of SEQ ID NO:3, (b) a mutation in the BBLd-1polynucleotide sequence having at least 97% identity to the nucleotidesequence of SEQ ID NO:1 (BBLd-1), and/or (c) a mutation in the BBLd-2polynucleotide sequence having at least 97% identity to the nucleotidesequence of SEQ ID NO:2 (BBLd-2). In some embodiments, the nicotinicalkaloid comprises, consists essentially of, or consists of nicotine.

Procedures for determining nicotinic alkaloid content are well known androutine in the art and are described throughout the literature.Non-limiting examples of such methods include gas chromatography, massspectrometry (Domino et al. 1992 Med Sci Res. 20:859-860; Sheen et al.2006 J Food Sci 53(5):1572-1573), HPLC (Keinänen et al. 2001 J AgricFood Chem 49:3553-3558; Halitschke and Baldwin 2003 Plant J 36:794-807), UV absorption (Willits et al. 2005 Analytical Chemistry22:430-433), and the like.

In some embodiments, a mutation may reduce or eliminate expression of(a) a BBLe polynucleotide having 97% identity to the nucleotide sequenceof SEQ ID NO:3, (b) a BBLd-1 polynucleotide having 97% identity to thenucleotide sequence of SEQ ID NO:1, and/or (c) a BBLd-2 polynucleotidehaving 97% identity to the nucleotide sequence of SEQ ID NO:2. In someembodiments, a mutation may reduce or eliminate activity of (a) apolypeptide encoded by the BBLe polynucleotide having 97% identity tothe nucleotide sequence of SEQ ID NO:3, (b) a polypeptide encoded by theBBLd-1 polynucleotide having 97% identity to the nucleotide sequence ofSEQ ID NO:1, and/or (c) a polypeptide encoded by the BBLd-2polynucleotide having 97% identity to the nucleotide sequence of SEQ IDNO:2. In representative embodiments, expression of a BBLe, BBLd-1,BBLd-2 polynucleotide and/or the activity of a BBLe, BBLd-1, BBLd-2polypeptide may be reduced by at least about 30% (e.g., about 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or anyrange or value therein).

Introducing a mutation may comprise, consist essentially of, or consistof in planta modification of one or more of the wild-type or nativenucleotide sequences encoding the BBL polynucleotides of this invention(e.g., a BBLe polynucleotide having 97% identity to the nucleotidesequence of SEQ ID NO:3, a BBLd-1 polynucleotide having 97% identity tothe nucleotide sequence of SEQ ID NO:1, and/or (c) a BBLd-2polynucleotide having 97% identity to the nucleotide sequence of SEQ IDNO:2). Any method of modify a nucleotide sequence in planta can be usedwith this invention to alter the expression of the genes encoding theseBBL polynucleotides. Thus, in some embodiments, introducing a mutationinto a Nicotiana plant may comprise, consist essentially of, or consistof chemical mutagenesis, insertional mutagenesis and/or irradiation ofthe plant or a plant part. Non-limiting examples of chemical mutagensinclude ethylmethane sulfonate (EMS), nitrosoguanidine, bisulfite,N4-hydroxycytidine, and/or aminopurine. Non-limiting examples ofirradiation mutagens include gamma-ray, x-ray, and/or fast neutronirradiation. Insertional mutagenesis techniques include but are notlimited to T-DNA tagging, transposon tagging and mutagenicoligonucleotides. In representative embodiments, the mutation may be adeletion or an insertion, optionally a deletion or insertion thatresults in a net loss or gain of nucleotides that is not divisible bythree, thereby generating a frameshift mutation that may result in anonfunctional protein, if a protein is produced at all.

In some embodiments, introducing the mutation into a Nicotiana plant orplant part comprises introducing into the Nicotiana plant or plant partat least one recombinant nucleic acid encoding a nuclease targeting (a)a BBLe polynucleotide having 97% identity to the nucleotide sequence ofSEQ ID NO:3, (b) a BBLd-1 polynucleotide having 97% identity to thenucleotide sequence of SEQ ID NO:1, and/or (b) a BBLd-2 polynucleotidehaving 97% identity to the nucleotide sequence of SEQ ID NO:2. In someembodiments, the introduction of the recombinant nucleic acid can bestable or transient. In some embodiments, when the introduction isstable, the introduced constructs (e.g., the at least one recombinantnucleic acid) can be removed, for example, by crossing the transformedplant with non-modified plants or through segregation of non-homozygousplants. Non-limiting examples of a nuclease useful for generatingmutants in an endogenous BBL polynucleotide of the invention includes ameganuclease, a zinc finger nuclease (ZFN), a transcriptionactivator-like effector nuclease (TALEN), and/or a clustered regularlyinterspaced, short palindromic repeat (CRISPR) associated (Cas)nuclease. Use of such nucleases for creating targeted mutations is wellknown in the art and routine. In representative embodiments, themutation may be a deletion or an insertion, optionally a deletion orinsertion resulting in a net loss or gain of nucleotides that is notdivisible by three, thereby generating a frameshift mutation that mayresult in a nonfunctional protein, if a protein is produced at all.

Meganucleases, endodeoxyribonucleases, or homing enzymes compriserecognition sites of from about 12-40 base pairs of double stranded DNA.These large recognition sites make these nucleases very specific fortheir target site. Exemplary meganucleases useful in genome modificationinclude I-SceI, I-DmoI, and I-CreI. In some embodiments, a meganucleasemay be modified to alter the portion of the amino acid sequence of themeganuclease that recognizes the target DNA, thereby generatingmeganucleases that recognize different DNA sequences (see, e.g., U.S.Pat. No. 8,021,867). Use of meganucleases for targeted DNA modificationof gene sequences is well known in the art (see, Stoddard, B. L.Quarterly Reviews of Biophysics 38(1):49-95 (2006). doi:10.1017/S0033583505004063; Amould et al., Journal of Molecular Biology355(3):443-58 (2006). doi:10.1016/j.jmb.2005.10.065; Delacôte et al.PloS One 8(1):e53217 (2013 Jan. 1). doi:10.1371/journal.pone.0053217; deSouza, N., Nat Meth 9 (1), 27-27 (2011); and Antunes et al. BMCBiotechnology 12:86 (2012)). Thus, in some embodiments, when thenuclease is a meganuclease, the target region can be about 12 to about40 consecutive base pairs in length (e.g., about 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40 consecutive base pairs, or any range or valuetherein) from a BBLe polynucleotide having 97% identity to thenucleotide sequence of SEQ ID NO:3, a BBLd-1 polynucleotide having 97%identity to the nucleotide sequence of SEQ ID NO:1, or a BBLd-2polynucleotide having 97% identity to the nucleotide sequence of SEQ IDNO:2. In exemplary embodiments, the target region can be about 14 to 40consecutive base pairs in length, about 15 to about 35 consecutive basepairs in length, about 18 to about 30 consecutive base pairs in length,or about 20 to about 30 consecutive base pairs in length. In particularembodiments, the target region can be about 22 consecutive base pairs inlength. In some embodiments, the nuclease can be I-CreI meganuclease.

Zinc finger nucleases (ZFNs) are chimeric proteins comprising at leastone zinc finger DNA binding domain linked to at least one nucleasecapable of cleaving DNA. Exemplary nucleases useful in a ZFN are FokIrestriction enzymes. (Urnov et al. Nature Reviews Genetics 11, 636-646(2010)). Zinc finger binding domains can be “engineered” to bind to apredetermined nucleotide sequence (See, for example, Id., U.S. Pat. Nos.6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059;WO 98/53060; WO 02/016536 and WO 03/016496. Cleavage by a ZFN at atarget nucleic acid typically results in a double stranded break at thetarget site. Notably, ZFNs are unique in that their DNA target siteswill always be divisible by 3. In some embodiments, the DNA target sitecan range from about 9 consecutive base pairs to about 30 consecutivebase pairs in length (e.g., about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 base pairs in length).Accordingly, in some embodiments, the target site for a ZFN can be about9 consecutive base pairs to about 30 consecutive base pairs (or anyrange or value therein) from a BBLe polynucleotide having 97% identityto the nucleotide sequence of SEQ ID NO:3, a BBLd-1 polynucleotidehaving 97% identity to the nucleotide sequence of SEQ ID NO:1, or aBBLd-2 polynucleotide having 97% identity to the nucleotide sequence ofSEQ ID NO:2

Transcription activator-like effector nucleases (TALENs) are chimericproteins comprising FokI nucleases fused with transcription activatorlike proteins (TAL) (i.e., TAL effector DNA binding domain). TALproteins are composed of 33-35 amino acid repeating motifs with variablepositions that have a strong recognition for specific nucleotides (Denget al. Protein Cell 5(4):297-306 (2014)) (approximately 10-30 repeatunits per binding domain). One specific DNA base is recognized through ahighly variable residue at a fixed position in the TAL repeat. EachTALEN fusion thus provides a TALEN monomer consisting of a TAL effectorDNA binding domain with a Fok1 catalytic domain fused to its C terminus.Since FokI cleaves as a dimer, the TAL effector nucleases function inpairs, binding opposing DNA target sites across a gap located betweenthe target sites over which the FokI domains come together to create thebreak. Thus, a pair of TALENs bind to candidate target sites orientedfrom 5′ to 3′ on opposite strands of DNA having a spacer region betweenthe sites, wherein the spacer region is large enough for the two Fok1domains to dimerize and cut the DNA, but not so large that the two Fok1domains do not come into contact (Cermak et al. Nucleic Acids Res.39(12): e82 (2011)). Methods for making and using TALENs for DNAmodification are well known and routine in the art (see, e.g., U.S. Pat.Nos. 8,440,432; 8,507,272; 8,912,138; Miller, J. C. et al. Nat.Biotechnol. 29, 143-148 (2011); and Christian, M. et al. Genetics 186,757-761 (2010)). In some embodiments, a DNA target site for TALEN canrange from about 9 consecutive base pairs to about 40 consecutive basepairs in length (e.g., about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40 base pairs in length). Accordingly, in some embodiments, thetarget site for a ZFN can be about 9 consecutive base pairs to about 40consecutive base pairs (or any range or value therein) from a BBLepolynucleotide having 97% identity to the nucleotide sequence of SEQ IDNO:3, a BBLd-1 polynucleotide having 97% identity to the nucleotidesequence of SEQ ID NO:1, or a BBLd-2 polynucleotide having 97% identityto the nucleotide sequence of SEQ ID NO:2.

Clustered regularly interspaced, short palindromic repeat (CRISPR)associated (Cas) nucleases are also useful for modifying endogenous BBLpolynucleotides. In contrast to TALENS, ZFNs and homing meganucleases,newly engineered CRISPR-Cas nucleases are not required for each use. Therequirements for cleavage and DNA modification using CRISPR-Cas systemsare well known and routine. (U.S. Pat. Nos. 8,906,616; 8,895,308;8,993,233; Jiang et al. 2013. Nat. Biotechnol. 31:233-239; Briner et al.Mol. Cell 56:333-339 (2014); Cong et al. Science 339:819-823 (2013);Gilbert et al. Cell 154: 442-451 (2013); Jinek et al. Science337:816-821 (2012); Qi et al. Cell 152:1173-1183 (2013); Ran et al.Nature Protocols 8:2281-2308 (2013); and Sander et al. Nat. Biotechnol32, 347-355 (2014)). Type II CRISPR systems are the most commonly usedfor genome editing. The bacterial Type II CRISPR systems comprise twoRNA components, a CRISPR RNA (crRNA) and a transactivating RNA(tracrRNA). To facilitate use in genome engineering applications, thesetwo RNA components may be combined into a single RNA that is referred toas a guide RNA (gRNA or sgRNA). The most commonly used genomeengineering system is the CRISPR-Cas9 system derived from Streptococcuspyogenes.

For successful cleavage, a guide RNA is designed to comprise a“recognition motif” that is complementary to a target DNA sequence(protospacer) that is next to (e.g., immediately downstream of) a“protospacer adjacent motif” (PAM) sequence. In some embodiments, thePAM sequence may be NGG. In some embodiments, a recognition motif of agRNA for a CRISPR-Cas system can comprise a sequence having length ofabout 10 consecutive base pairs to about 30 consecutive base pairs(e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30 consecutive base pairs, and any range or valuetherein) that is complementary to a target DNA (protospacer) (e.g., to aBBLe polynucleotide having 97% identity to the nucleotide sequence ofSEQ ID NO:3, a BBLd-1 polynucleotide having 97% identity to thenucleotide sequence of SEQ ID NO:1, or a BBLd-2 polynucleotide having97% identity to the nucleotide sequence of SEQ ID NO:2). In someembodiments, the spacer comprises a length of about 15 to about 30,about 15 to about 25, about 15 to about 20, about 18 to about 30, about18 to about 25, about 18 to about 20, about 20 to about 30, about 25 toabout 30, and the like, consecutive base pairs having complementarity toa target DNA. In some embodiments, two gRNA constructs positioned inclose proximity and opposite orientation (like the two monomers of aTALENs) may be generated and used with Cas9 nucleases having a singlenickase activity. Such a design may increase target specificity. In someembodiments, the nickase activity of the Cas9 may be inactivated and theCas9 fused to Fok1 nuclease (fCas9) so that the Fok1 nuclease onlyfunctions when two fCas9 “monomer” units dimerize. Again, such a designmay increase target specificity. (Guilinger et al. Nature Biotechnol.32:577-588(2014))

In some embodiments, the invention further comprises reducing theactivity of at least one additional nicotinic alkaloid biosyntheticenzyme and/or reducing the expression of a polynucleotide encoding atleast one additional nicotinic alkaloid biosynthetic enzyme in aNicotiana plant or plant part. Thus, in some embodiments, in addition tointroducing into a Nicotiana plant or plant part a mutation in at leastone of an endogenous BBLe polynucleotide having 97% identity to thenucleotide sequence of SEQ ID NO:3, a mutation in an endogenous BBLd-1polynucleotide having 97% identity to the nucleotide sequence of SEQ IDNO:1, and/or a mutation in an endogenous BBLd-2 polynucleotide having97% identity to the nucleotide sequence of SEQ ID NO:2, the Nicotianaplant or plant part may be further modified so as to reduce the activityof additional nicotinic alkaloid biosynthetic enzymes or reduce theexpression of nucleic acids encoding the additional nicotinic alkaloidbiosynthetic enzymes. Such additional nicotinic alkaloid biosyntheticenzymes include but are not limited to additional berberine bridgeenzyme-like polypeptide (e.g., BBLa, BBLb, BBLc), aspartate oxidase,quinolinate synthase, quinolate phosphoribosyl transferase, ornithinedecarboxylase, putrescine N-methyltransferase, methyl putrescineoxidase, and A622. Thus, for example, in addition to introducing into aNicotiana plant or plant part a mutation in at least one of anendogenous BBLe polynucleotide having 97% identity to the nucleotidesequence of SEQ ID NO:3, a mutation in an endogenous BBLd-1polynucleotide having 97% identity to the nucleotide sequence of SEQ IDNO:1, and/or a mutation in an endogenous BBLd-2 polynucleotide having97% identity to the nucleotide sequence of SEQ ID NO:2, the Nicotianaplant or plant part may be further modified so as to reduce theexpression of BBLa, BBLb, and/or BBLc and/or reduce the activity of anadditional berberine bridge enzyme-like polypeptide such as BBLa, BBLb,and/or BBL. In some embodiments, in addition to introducing into aNicotiana plant or plant part a mutation in at least one of anendogenous BBLe polynucleotide having 97% identity to the nucleotidesequence of SEQ ID NO:3, a mutation in an endogenous BBLd-1polynucleotide having 97% identity to the nucleotide sequence of SEQ IDNO:1, and/or a mutation in an endogenous BBLd-2 polynucleotide having97% identity to the nucleotide sequence of SEQ ID NO:2, the Nicotianaplant or plant part may be further modified to reduce the expression ofBBLa, BBLb, and/or BBLc and/or reduce the activity of an additionalberberine bridge enzyme-like polypeptide such as BBLa, BBLb, and/or BBL,wherein the modification to reduce the expression of BBLa, BBLb, and/orBBLc or the activity of BBLa, BBLb, and/or BBL comprises, consistsessentially of, or consists of an ethyl methanesulfonate (EMS) mutationof a nucleotide sequence encoding BBLa, BBLb, and/or BBLc.

In some embodiments, the invention further comprises reducing expressionof a polynucleotide encoding a transcription factor that positivelyregulates nicotinic alkaloid biosynthesis in a Nicotiana plant or plantpart. Thus, in some embodiments, in addition to introducing into aNicotiana plant or plant part a mutation in at least one of anendogenous BBLe polynucleotide having 97% identity to the nucleotidesequence of SEQ ID NO:3, a mutation in an endogenous BBLd-1polynucleotide having 97% identity to the nucleotide sequence of SEQ IDNO:1, and/or a mutation in an endogenous BBLd-2 polynucleotide having97% identity to the nucleotide sequence of SEQ ID NO:2, a Nicotianaplant or plant part may be further modified to reduce expression of atleast one polynucleotide encoding a transcription factor that positivelyregulates nicotinic alkaloid biosynthesis. Non-limiting examples oftranscription factors that positively regulate nicotinic alkaloidbiosynthesis include ERF family transcription factors such as ERF189,ERF221 and ERF32, and/or bHLH family transcription factors such asNtMYC1 and NtMYC2, and COI1.

In some embodiments, the invention further comprises overexpression ofat least one polynucleotide encoding a transcription factor thatnegatively regulates nicotinic alkaloid biosynthesis in a Nicotianaplant or plant part. Thus, in some embodiments, in addition tointroducing into a Nicotiana plant or plant part a mutation in at leastone of an endogenous BBLe polynucleotide having 97% identity to thenucleotide sequence of SEQ ID NO:3, a mutation in an endogenous BBLd-1polynucleotide having 97% identity to the nucleotide sequence of SEQ IDNO:1, and/or a mutation in an endogenous BBLd-2 polynucleotide having97% identity to the nucleotide sequence of SEQ ID NO:2, a Nicotianaplant or part thereof may be further modified to overexpress at leastone polynucleotide encoding a transcription factor that negativelyregulates nicotinic alkaloid biosynthesis. Non-limiting examples oftranscription factors that negatively regulate nicotinic alkaloidbiosynthesis includes JAZ.

As used herein, “overexpress,” “overexpression,” overexpressed, (andgrammatical variations thereof) refer to the production of a geneproduct in a transgenic Nicotiana plant or plant part that exceeds thelevel of production of the same gene product in a control Nicotianaplant or plant part, the transgenic Nicotiana plant or plant part beingtransformed with a recombinant nucleic acid construct that confers theincreased production of the gene product, whereas the control Nicotianaplant or plant part is not transformed with said recombinant nucleicacid construct.

The expression and activity of any of additional polynucleotides to bealtered in a Nicotiana plant or plant part of this invention may bereduced by introducing mutations as described herein for BBLe, BBLd-1,BBLd-2, or may be reduced by other well known means of reducingexpression of polynucleotides and activity of polypeptides including butnot limited to use of interfering RNAs developed from the nucleic acidsencoding the additional nicotinic alkaloid biosynthetic enzymes. As iswell known in the art, “interfering RNA” is RNA capable of causing genesilencing. Interfering RNA, as used herein, includes any type of RNAmolecule capable of down-regulating or silencing expression of a targetnicotinic alkaloid biosynthetic nucleic acid, including but not limitedto sense RNA, antisense RNA, short interfering RNA (siRNA), microRNA(miRNA), double-stranded RNA (dsRNA), hairpin RNA (RNA) and the like.

In additional embodiments, the invention further provides a Nicotianaplant and/or plant part comprising reduced nicotinic alkaloid contentproduced by any of the methods of the invention. In some embodiments,the Nicotiana plant part may be a Nicotiana plant cell. Thus, in someembodiments, the invention provides a Nicotiana plant or plant part ofthe invention having reduced nicotinic alkaloid content and comprising,consisting essentially of, or consisting of a mutation in (a) a BBLepolynucleotide having 97% identity to the nucleotide sequence of SEQ IDNO:3, (b) a BBLd-1 polynucleotide having 97% identity to the nucleotidesequence of SEQ ID NO:1, and/or (c) a BBLd-2 polynucleotide having 97%identity to the nucleotide sequence of SEQ ID NO:2. In some embodiments,the mutation may be a deletion or insertion mutation. In someembodiments, the expression of the BBLe polynucleotide having 97%identity to the nucleotide sequence of SEQ ID NO:3 may be reduced byabout 30% to about 100%, the expression of the BBLd-1 polynucleotidehaving 97% identity to the nucleotide sequence of SEQ ID NO:1 may bereduced by about 30% to about 100%, and/or the expression of the BBLd-2polynucleotide having 97% identity to the nucleotide sequence of SEQ IDNO:2 may be reduced by about 30% to about 100%. In some embodiments, theactivity of a polypeptide produced by the BBLe polynucleotide having 97%identity to the nucleotide sequence of SEQ ID NO:3 may be reduced byabout 30% to about 100%, the activity of a polypeptide produced by theBBLd-1 polynucleotide having 97% identity to the nucleotide sequence ofSEQ ID NO:1 may be reduced by about 30% to about 100%, and/or theactivity of a polypeptide produced by the BBLd-2 polynucleotide having97% identity to the nucleotide sequence of SEQ ID NO:2 may be reduced byabout 30% to about 100%. In some embodiments, the Nicotiana plant orplant part may further comprise (1) reduced activity and/or reducedexpression of at least one additional nicotinic alkaloid biosyntheticpolypeptide or polynucleotide, respectively, and/or (2) reducedexpression of a encoding a transcription factor that positivelyregulates nicotinic alkaloid biosynthesis, and/or (3) increasedexpression (overexpression) of a encoding a transcription factor thatnegatively regulates nicotinic alkaloid biosynthesis.

In some embodiments, the nicotine content of a Nicotiana plant or plantpart of the invention (e.g., having at least reduced nicotinic alkaloidcontent and comprising a mutation in (a) a BBLe polynucleotide having97% identity to the nucleotide sequence of SEQ ID NO:3, (b) a BBLd-1polynucleotide having 97% identity to the nucleotide sequence of SEQ IDNO:1, and/or (c) a BBLd-2 polynucleotide having 97% identity to thenucleotide sequence of SEQ ID NO:2) can be about 10 mg/g (e.g., about1%) to about 100 mg/g (e.g., 10%) (e.g., 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100 mg/g, and any range or value therein) dryweight nicotine. In particular embodiments, the nicotine content of aNicotiana plant or plant part of the invention can be about 20 mg/g(e.g., about 2%) to about 100 mg/g (e.g., 10%) dry weight nicotine;about 30 mg/g to about 100 mg/g dry weight nicotine, about 40 mg/g toabout 100 mg/g dry weight nicotine, about 50 mg/g to about 100 mg/g dryweight nicotine, and the like. In still other embodiments, the nicotinecontent of a Nicotiana plant or plant part of the invention can be atleast about 30 mg/g dry weight nicotine, at least about 40 mg/g (e.g.,about 4%) dry weight nicotine, at least about 50 mg/g (e.g., about 5%)dry weight nicotine, at least about 60 mg/g (e.g., about 6%) dry weightnicotine, and the like. In some embodiments, the nicotine content can bereduced to less than 10 mg/g (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0mg/g nicotine)

In some embodiments, a Nicotiana seed of a plant of the invention andNicotiana plants produced from the seed are provided, wherein the seedcomprises a mutation in the BBLe polynucleotide having 97% identity tothe nucleotide sequence of SEQ ID NO:3, a mutation in the BBLd-1polynucleotide having 97% identity to the nucleotide sequence of SEQ IDNO:1, and/or a mutation in the BBLd-2 polynucleotide having 97% identityto the nucleotide sequence of SEQ ID NO:2.

In some embodiments, the invention provides a progeny Nicotiana plantproduced from the Nicotiana plants of the invention. In someembodiments, further provided is a crop comprising a plurality ofNicotiana plants of the invention planted together in an agriculturalfield.

Additional aspects of the invention include a harvested product producedfrom the Nicotiana plants or plant parts of the invention, as well as aprocessed product produced from said harvested product. A harvestedproduct can be a whole plant or any plant part, wherein said harvestedproduct comprises a recombinant nucleic acid molecule/construct of theinvention. Thus, in some embodiments, non-limiting examples of aharvested product include a seed, a fruit, a flower or part thereof(e.g., an anther, a stigma, and the like), a leaf, a stem, and the like.

In some embodiments, the present invention provides a tobacco product,wherein the product can be a blended tobacco product. In otherembodiments of the invention, the tobacco product of the presentinvention can be a reduced nicotine tobacco product. In still otherembodiments, the tobacco product of the present invention can be ablended tobacco product with reduced nicotine content. Thus, the tobaccoproduct of the present invention can be a blended reduced nicotinetobacco product.

In some embodiments, a tobacco product may include, but is not limitedto, leaf tobacco, shredded tobacco, cut tobacco, ground tobacco, powdertobacco, tobacco extract, smokeless tobacco, moist or dry snuff, kretek,pipe tobacco, cigar tobacco, cigarillo tobacco, cigarette tobacco,chewing tobacco, cigarettes, cigars, bidis, bits, and tobacco-containinggum and lozenges. In particular embodiments, the tobacco product may bea cigarillo, a non-ventilated recess filter cigarette, a vented recessfilter cigarette, a cigar, snuff, and chewing tobacco. In someembodiments, the tobacco product may be produced from a form of tobaccoincluding but not limited to leaf tobacco, shredded tobacco, cuttobacco, ground tobacco, powder tobacco, tobacco extract, and anycombination thereof. In some embodiments, a tobacco product of theinvention comprises a blended tobacco product, a reduced nicotinetobacco product, and any combination thereof.

Those of skill in the art understand that tobacco plants, or partsthereof, are traditionally cured prior to use in a tobacco product.Thus, additional embodiments of the present invention include tobaccoproducts comprising cured tobacco made from a Nicotiana plant of theinvention having reduced nicotinic alkaloid content and comprising amutation in (a) a BBLe polynucleotide having 97% identity to thenucleotide sequence of SEQ ID NO:3, (b) a BBLd-1 polynucleotide having97% identity to the nucleotide sequence of SEQ ID NO:1, and/or (c) aBBLd-2 polynucleotide having 97% identity to the nucleotide sequence ofSEQ ID NO:2. Accordingly, tobacco plants and plant parts can be curedaccordingly to processes known to those of skill in the art. Suchprocesses include, but are not limited to, air curing, ground curing,rack curing, pit curing, fire curing, sun curing and flue curing.

The present invention further provides a method of producing a tobaccoproduct, comprising providing a cured tobacco, wherein the cured tobaccocomprises the leaves of a Nicotiana plant of the invention havingreduced nicotinic alkaloid content and comprising a mutation in (a) aBBLe polynucleotide having 97% identity to the nucleotide sequence ofSEQ ID NO:3, (b) a BBLd-1 polynucleotide having 97% identity to thenucleotide sequence of SEQ ID NO:1, and/or (c) a BBLd-2 polynucleotidehaving 97% identity to the nucleotide sequence of SEQ ID NO:2; andproducing said tobacco product from said cured tobacco.

In some embodiments, the invention provides a reduced-nicotinic alkaloidtobacco product produced from a Nicotiana plant or plant part of theinvention having reduced nicotinic alkaloid content and comprising amutation in (a) a BBLe polynucleotide having 97% identity to thenucleotide sequence of SEQ ID NO:3, (b) a BBLd-1 polynucleotide having97% identity to the nucleotide sequence of SEQ ID NO:1, and/or (c) aBBLd-2 polynucleotide having 97% identity to the nucleotide sequence ofSEQ ID NO:2.

The present invention further provides a method of producing a blendedtobacco, comprising: a) providing a first tobacco; b) providing a secondtobacco, wherein the second tobacco is produced from a Nicotiana plantof the invention having reduced nicotinic alkaloid content andcomprising a mutation in (a) a BBLe polynucleotide having 97% identityto the nucleotide sequence of SEQ ID NO:3, (b) a BBLd-1 polynucleotidehaving 97% identity to the nucleotide sequence of SEQ ID NO:1, and/or(c) a BBLd-2 polynucleotide having 97% identity to the nucleotidesequence of SEQ ID NO:2; and c) blending said first tobacco with saidsecond tobacco so as to produce said blended tobacco. In someembodiments, the first and the second tobacco may be produced from aNicotiana plant of the invention, wherein, in some embodiments, thefirst tobacco and second tobacco are from different Nicotiana plantvarieties both having reduced nicotinic alkaloid content and comprisinga mutation in (a) a BBLe polynucleotide having 97% identity to thenucleotide sequence of SEQ ID NO:3, (b) a BBLd-1 polynucleotide having97% identity to the nucleotide sequence of SEQ ID NO:1, and/or (c) aBBLd-2 polynucleotide having 97% identity to the nucleotide sequence ofSEQ ID NO:2.

In other aspects of the present invention, a method is provided forproducing a blended reduced nicotine tobacco, the method comprising: a)providing a first tobacco; b) providing a second tobacco, wherein thesecond tobacco is produced from a Nicotiana plant of the inventionhaving reduced nicotinic alkaloid content and comprising a mutation in(a) a BBLe polynucleotide having 97% identity to the nucleotide sequenceof SEQ ID NO:3, (b) a BBLd-1 polynucleotide having 97% identity to thenucleotide sequence of SEQ ID NO:1, and/or (c) a BBLd-2 polynucleotidehaving 97% identity to the nucleotide sequence of SEQ ID NO:2; and c)blending said first tobacco with said second tobacco so as to producesaid blended reduced nicotine tobacco. In some embodiments, first andthe second tobacco may be produced from a Nicotiana plant of theinvention, wherein, in some embodiments, the first tobacco and secondtobacco are from different Nicotiana plant varieties both having reducednicotinic alkaloid content and comprising a mutation in (a) a BBLepolynucleotide having 97% identity to the nucleotide sequence of SEQ IDNO:3, (b) a BBLd-1 polynucleotide having 97% identity to the nucleotidesequence of SEQ ID NO:1, and/or (c) a BBLd-2 polynucleotide having 97%identity to the nucleotide sequence of SEQ ID NO:2. As an example, bothflue-cured and air-cured tobaccos are components of the common Americanblend cigarette. Thus, in some embodiments of the invention, a lownicotinic alkaloid tobacco product may be produced by blending a lownicotine burley variety with a high nicotine flue-cured variety, eachvariety having reduced nicotinic alkaloid content and comprising amutation in (a) a BBLe polynucleotide having 97% identity to thenucleotide sequence of SEQ ID NO:3, (b) a BBLd-1 polynucleotide having97% identity to the nucleotide sequence of SEQ ID NO:1, and/or (c) aBBLd-2 polynucleotide having 97% identity to the nucleotide sequence ofSEQ ID NO:2, thereby reducing the overall nicotinic alkaloid (e.g.,nicotine, anatabine, nornicotine, anabasine, and the like) content ofthe low alkaloid tobacco product.

As is well known in the art, a tobacco formulation for a tobacco productcan incorporate other components in addition to tobacco which can alterthe bitterness, sweetness, sourness or saltiness of the formulation;enhance the perceived dryness or moistness of the formulation; or thedegree of tobacco taste exhibited by the formulation. Such othercomponents may be salts (e.g., sodium chloride, potassium chloride,sodium citrate, potassium citrate, sodium acetate, potassium acetate,and the like); natural sweeteners (e.g., fructose, sucrose, glucose,maltose, mannose, galactose, lactose, and the like); artificialsweeteners (e.g., sucralose, saccharin, aspartame, acesulfame K, and thelike), organic and inorganic fillers (e.g., grains, processed grains,puffed grains, maltodextrin, dextrose, calcium carbonate, calciumphosphate, corn starch, lactose, manitol, xylitol, sorbitol, finelydivided cellulose, and the like); binders (e.g., povidone, sodiumcarboxymethylcellulose and other modified cellulosic types of binders,sodium alginate, xanthan gum, starch-based binders, gum arabic,lecithin, and the like); pH adjusters or buffering agents (e.g., metalhydroxides, preferably alkali metal hydroxides such as sodium hydroxideand potassium hydroxide, and other alkali metal buffers such aspotassium carbonate, sodium carbonate, sodium bicarbonate, and thelike); colorants (e.g., dyes and pigments, including caramel coloringand titanium dioxide, and the like); humectants (e.g. glycerin,propylene glycol, and the like); preservatives (e.g., potassium sorbate,and the like); syrups (e.g., honey, high fructose corn syrup, and thelike); disintegration aids (e.g., microcrystalline cellulose,croscarmellose sodium, crospovidone, sodium starch glycolate,pregelatinized corn starch, and the like); antioxidants (e.g., ascorbicacid, grape seed extracts and oils, polyphenol-containing materials suchas green tea extract and black tea extract, peanut endocarb, potatopeel, and the like (See Santhosh et al., Phytomedicine, 122:16-220(2005); incorporated herein by reference); and flavoring agents.Flavoring agents may be natural or synthetic, and include, but are notlimited to, fresh, sweet, herbal, confectionary, floral, fruity orspice. Specific types of flavors include, but are not limited to,vanilla, coffee, chocolate, cream, mint, spearmint, menthol, peppermint,wintergreen, lavender, cardamon, nutmeg, cinnamon, clove, cascarilla,sandalwood, honey, jasmine, ginger, anise, sage, licorice, grape, lemon,orange, apple, peach, lime, cherry, and strawberry. (See Leffingwill etal., Tobacco Flavoring for Smoking Products, R. J. Reynolds TobaccoCompany (1972)). Flavorings also can include components that areconsidered moistening, cooling or smoothening agents, including, but notlimited to, eucalyptus. These flavors may be provided alone or in acomposite (e.g., spearmint and menthol, or orange and cinnamon).Representative types of components are also set forth in U.S. Pat. No.5,387,416 to White et al. and PCT Application Publication No. WO2005/041699 to Quinter et al., the relevant portions of each of which isincorporated herein by reference. Thus, in some embodiments, the tobaccoproduct of the invention may comprise a flavoring component or a scent.

The amount of tobacco within the tobacco formulation may vary. Inparticular embodiments, the amount of tobacco within the tobaccoformulation is at least about 25 percent to at least about 40 percent,on a dry weight basis (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40% dry weight, and any value or range therein). Theamounts of other components within the tobacco formulation preferablyare in excess of about 25 percent to about 40 percent, on a dry weightbasis.

In some embodiments of the present invention, methods are providedwherein the amount of nicotine in a human that uses tobacco is reduced,the method comprising providing to said human any of the tobaccoproducts of the present invention.

In still other aspects of the present invention, a method is providedfor reducing the nicotine consumption of a tobacco user, the methodcomprising: (a) providing said tobacco user a first tobacco productcomprising tobacco produced from a Nicotiana plant of the inventionhaving reduced nicotinic alkaloid content and comprising a mutation in(a) a BBLe polynucleotide having 97% identity to the nucleotide sequenceof SEQ ID NO:3, (b) a BBLd-1 polynucleotide having 97% identity to thenucleotide sequence of SEQ ID NO:1, and/or (c) a BBLd-2 polynucleotidehaving 97% identity to the nucleotide sequence of SEQ ID NO:2; and (b)providing said tobacco user a second tobacco product comprising tobaccoproduced from a Nicotiana plant of the invention having reducednicotinic alkaloid content and comprising a mutation in (a) a BBLepolynucleotide having 97% identity to the nucleotide sequence of SEQ IDNO:3, (b) a BBLd-1 polynucleotide having 97% identity to the nucleotidesequence of SEQ ID NO:1, and/or (c) a BBLd-2 polynucleotide having 97%identity to the nucleotide sequence of SEQ ID NO:2; wherein said secondtobacco product comprises less nicotine than said first tobacco product.

In some aspects of the invention, a tobacco user can be provided withadditional tobacco products comprising tobacco produced from a Nicotianaplant of the invention having reduced nicotinic alkaloid content andcomprising a mutation in (a) a BBLe polynucleotide having 97% identityto the nucleotide sequence of SEQ ID NO:3, (b) a BBLd-1 polynucleotidehaving 97% identity to the nucleotide sequence of SEQ ID NO:1, and/or(c) a BBLd-2 polynucleotide having 97% identity to the nucleotidesequence of SEQ ID NO:2; wherein said additional tobacco productscomprise sequentially reduced amounts of nicotine, starting with a thirdproduct that comprises less nicotine than said first or second tobaccoproduct.

In other embodiments of the present invention tobacco-use cessation kitsare provided, wherein the tobacco-use cessation kits comprise a tobaccoproduct selected from the tobacco products of any of the products of thepresent invention produced from a Nicotiana plant of the inventionhaving reduced nicotinic alkaloid content and comprising a mutation in(a) a BBLe polynucleotide having 97% identity to the nucleotide sequenceof SEQ ID NO:3, (b) a BBLd-1 polynucleotide having 97% identity to thenucleotide sequence of SEQ ID NO:1, and/or (c) a BBLd-2 polynucleotidehaving 97% identity to the nucleotide sequence of SEQ ID NO:2.

In still other embodiments, the present invention provides a kitcomprising a first tobacco product that comprises nicotine and a secondtobacco product that comprises an amount of nicotine less than theamount in the first tobacco product, wherein said first or secondtobacco product comprises a tobacco product produced from a Nicotianaplant of the invention having reduced nicotinic alkaloid content andcomprising a mutation in (a) a BBLe polynucleotide having 97% identityto the nucleotide sequence of SEQ ID NO:3, (b) a BBLd-1 polynucleotidehaving 97% identity to the nucleotide sequence of SEQ ID NO:1, and/or(c) a BBLd-2 polynucleotide having 97% identity to the nucleotidesequence of SEQ ID NO:2.

In yet other embodiments, the present invention provides a productproduced from a Nicotiana plant of the invention having reducednicotinic alkaloid content and comprising a mutation in (a) a BBLepolynucleotide having 97% identity to the nucleotide sequence of SEQ IDNO:3, (b) a BBLd-1 polynucleotide having 97% identity to the nucleotidesequence of SEQ ID NO:1, and/or (c) a BBLd-2 polynucleotide having 97%identity to the nucleotide sequence of SEQ ID NO:2, wherein the productproduced is selected from the group consisting of industrial enzymes,pharmaceuticals, cosmetic components, human and livestock feeds, foodadditives, and fermentation products.

The invention will now be described with reference to the followingexamples. It should be appreciated that these examples are not intendedto limit the scope of the claims to the invention, but are ratherintended to be exemplary of certain embodiments. Any variations in theexemplified methods that occur to the skilled artisan are intended tofall within the scope of the invention.

EXAMPLES Example 1. BBL Mutations

To establish whether silencing the BBL gene family represents a viablemeans of obtaining reduced nicotine tobacco plants under conventionalfield growth conditions, we employed an RNAi approach (Lewis et al.,2015). Kajikawa et al., (2011) reported four unique BBL isoforms,designated BBLa, BBLb, BBLc and BBLd. To maximize the probability ofsuppressing the entire gene family, a 212 bp fragment from the mosthighly conserved region was selected. The anti-BBL RNAi construct wasgenerated specifically against the BBLa sequence because an in silicoanalysis of the tobacco EST sequences represented in GenBank suggestedthat it was the most highly expressed of the BBL isoforms. The 212 bpfragment from BBLa was 94%, 93% and 84% identical to the analogousregions of BBLb, BBLc and BBLd, respectively. Although the anti-BBL RNAiconstruct shared the least sequence identify with BBLd, both the insilico EST analysis and a reverse transcriptase PCR analysis conductedby Kajikawa et al. (2011) suggested that the BBLd isoform is minimallyexpressed compared to the other isoforms.

Flue-cured tobacco cultivar ‘K326’ was transformed with the anti-BBLRNAi construct and ten independent 35S:BBL-RNAi DH lines were selectedfor evaluation in replicated field experiments for alkaloid profiles,yield, and cured leaf quality. Six of the ten tested RNAi linesexhibited nicotine levels in cured leaf that were significantly lower(P<0.05) than that observed for the untransformed control line, K326(Table 1). The remaining four RNAi lines produced nicotine levels thatwere roughly equivalent to that of K326. This is likely because the RNAimechanism was not functioning in these transgenic lines. Transgenic lineDH32 exhibited the lowest percent nicotine in cured leaf (0.414%), whileuntransformed K326 produced 2.454% nicotine. The lowest nicotine level(0.299%) was produced by LAFC53, a nic1/nic1 nic2/nic2 isoline offlue-cured tobacco cultivar NC95.

TABLE 1 Means for measured characteristics for transgenic RNAi doubledhaploid lines and associated checks. Cured Leaf % % Cwt Cash % % TotalReduc- Yield Value Return % Nor- Anab- % % Alka- ing (kg ($ ($ GradeGenotype Nicotine nicotine asine Anatabine DMN loids Sugars ha⁻¹) cwt⁻¹)ha⁻¹) Index K326 Nbl-RNAi DH22A 0.5362 0.0784 0.0124 0.0143 0.01520.6565 13.71 1685 315.43 6439.64 83.3 K326 Nbl-RNAi DH32 0.4136 0.08900.0132 0.0073 0.0209 0.5440 12.67 1628 316.78 6551.81 83.7 K326 Nbl-RNAiDH16A 0.4720 0.0789 0.0125 0.0079 0.0163 0.5876 13.79 1439 309.344774.54 80.1 K326 Nbl-RNAi DH19 1.0442 0.0864 0.0178 0.0358 0.01651.2007 12.91 1615 322.99 5800.20 85.5 K326 Nbl-RNAi DH303 0.9110 0.08590.0166 0.0233 0.0178 1.0547 14.08 1944 311.13 7166.99 83.3 K326 Nbl-RNAiDH16B 0.7288 0.0781 0.0147 0.0283 0.0159 0.8658 13.32 1591 317.495831.29 84.0 K326 2.4541 0.0559 0.0206 0.1188 0.0104 2.6598 14.71 2343317.29 8254.99 83.9 NC95 2.8870 0.0636 0.0303 0.2217 0.0099 3.2125 12.951722 306.66 6105.61 81.3 LAFC53 0.2990 0.0128 0.0035 0.0201 0.00740.3428 8.55 1844 303.98 6519.67 79.4 LSD (0.05) 0.3998 0.0160 0.00320.0216 0.0039 0.4067 3.59 304 25.64 1098.66 6.4 % total alkaloids wascalculated as: % total alkaloids = (% nicotine + % nornicotine + %anatabine + % anabasine + % DMN).

All six of the RNAi lines with significantly lower percent nicotinerelative to K326 also exhibited significantly (P<0.05) lower anatabineand total alkaloid levels (Table 1) in the cured leaf. Five of these sixlines also exhibited significantly (P<0.05) lower levels of anabasine.All six were higher for percent nornicotine and percentdihydrometanicotine (DMN), although these numerical differences werevery small. The six RNAi lines exhibiting significantly lower nicotineand total alkaloid levels relative to K326 produced significantly lower(P<0.05) cured leaf yields as compared to untransformed K326, with anaverage difference of 693 kg ha⁻¹ (Table 1). No significant differenceswere detected, however, between K326 and any of the RNAi lines for curedleaf quality as measured by percent reducing sugars, value perhundredweight ($ cwt⁻¹), or grade index. In contrast, the LAFC53 plantshomozygous for nic1 and nic2 were clearly of lower quality, based ontheir overall lower grade index and reducing sugar content. Much of thequality reduction observed with LAFC53 was likely due to a delayedripening phenotype associated with this line when grown in the field.Overall, the results of this experiment demonstrate that inhibition ofBBL gene expression can give rise to tobacco plants with significantlyreduced nicotine while retaining important quality characteristics(Lewis et al., 2015).

Although RNAi suppression of BBL gene activity was effective inproducing high quality, reduced nicotine lines, there are many obstaclesassociated with the commercialization of a transgenic crop, such as theenormous costs and lengthy timelines associated with the deregulatoryprocess, as well as the fear of rejection by consumers who arephilosophically opposed to genetically modified (GM) crops. To provide anon-GM alternative, we screened an ethyl methane sulfonate(EMS)-mutagenized population of burley tobacco breeding line DH98 325-6for mutations in BBLa, BBLb and BBLc, the three most activelytranscribed genes of the BBL gene family. We did not screen formutations in BBLd because, as described above, this isoform appears tobe minimally expressed in comparison to the other three. Afterconducting thousands of high-throughput PCR and DNA sequencing reactionson this population, we were successful in identifying knockout(truncation) mutations in each of the three BBL genes that were screenedfor (Lopez, 2011; Lewis et al., 2015).

Sexual crossing in accompaniment with SNP genotyping was used to combinethe identified truncation mutations in BBLa, BBLb, and BBLc in allpossible homozygous combinations in the mutagenized DH98-325-6 geneticbackground. Alkaloid profiles were compared among plants of all sevenpossible bbl mutant genotypic classes as well as wild-type (WT)segregants grown in a single field environment (Table 2). Wide numericalranges were observed among the genotypic classes for percent nicotine,nornicotine, anatabine, and total alkaloids. Nornicotine was the mostprevalent alkaloid in all of these genotypes because DH98-325-6 has ahigh genetic potential to convert nicotine to nornicotine due to anactive nicotine demethylase gene designated as CYP82E4 (Lewis et al.,2010). Genotypes homozygous for single mutations exhibited slight tointermediate reductions in total alkaloids. Of the three single mutationgenotypes, the bbla/bbla mutation was found to have the largestnumerical effect, while the bblb/bblb mutation was found to have thesecond largest effect. The bblc/bblc mutation, by itself, provided onlya small reduction in percent total alkaloids. The double mutantcombination bbla/bbla bblb/bblb BBLc/BBLc and the triple homozygousmutation combination bbla/bbla bblb/bblb bblc/bblc exhibited the secondlowest and lowest levels of total alkaloid accumulation, respectively.These levels were substantially and significantly lower than that for WTDH98-325-6 segregants (Table 2). Although nornicotine was the majoralkaloid in these particular materials, once these mutations aretransferred to a normal, non-converter tobacco line, similar results areexpected with the exception that nicotine would represent thepredominant alkaloid species rather than nornicotine. These resultsconfirm that inactivation of BBL gene function represents an effectivemeans of producing low alkaloid tobacco plants, and that this can beaccomplished using a non-GM strategy (Lewis et al., 2015).

TABLE 2 Alkaloid content (% dry weight) of DH98 325-6 burley plantspossessing various combinations of BBL EMS-induced knockout mutations.Genotype Nic Nor Anab Anat TA NS BBLa/BBLb/BBLc 0.0907 1.4121 0.01090.0984 1.6121 SM BBLa/BBLb/bblc 0.1116 1.3256 0.0109 0.1136 1.5617 SMBBLa/bblb/BBLc 0.0885 0.9555 0.0090 0.0817 1.1347 SM bbla/BBLb/BBLc0.1086 0.9428 0.0111 0.0583 1.1208 DM BBLa/bblb/bblc 0.0471 1.18470.0119 0.1042 1.3478 DM bbla/BBLb/bblc 0.1054 1.1470 0.0139 0.09161.3579 DM bbla/bblb/BBLc 0.0056 0.3309 0.0126 0.0073 0.3564 TMbbla/bblb/bblc 0.0050 0.1001 0.0080 0.0058 0.1188 NS, null segregants(wild type); SM, single gene mutants; DM, double mutants; TM, triplemutants; TA, total alkaloid.

Example 2. Identification of New Members of the BBL Gene Family

Kajikawa et al. (2011) described the BBL gene family of tobacco as beingcomprised of four members, which they designated BBLa, BBLb, BBLc andBBLd. We conducted a BLAST-based analysis of the various databases foundin GenBank to determine whether there was evidence for the existence ofany other BBL family members within the tobacco genome. These resultsrevealed the presence of two additional BBL family members. One of theseshares 95% nucleotide identity and 94% predicted amino acid identitywith BBLd. Given that BBLd and this new isoform are very closely relatedto each other and are quite distinct from the other BBL genes (sharingless than 80% identity with any other gene family member), we refer tothese sequences as BBLd-1 (the original BBLd reported by Kajikawa etal., 2011) and BBLd-2 (the new isoform). The other novel BBL familymember that we identified shares 90-92% nucleotide identity with thepreviously characterized BBLa, BBLb and BBLc sequences. We designatethis new family member BBLe. The DNA and predicted protein sequences ofBBLe, BBLd-1 and BBLd-2 are shown below. Start and stop codons are inbold, underlined type. Locations of the 22 bp target sites of thecustom-designed meganuclease enzymes are highlighted grey.

BBLd-1 (same as GenBank accession #AB604221) SEQ ID NO: 1 ATGAAACGAAATATATCCATGTTTCTTCAGCTTCTGCTCATTATTCTGATGATGATCAGCTTCTTATTTACTTCTCTTCTTGTACCTTCGGTCTCTGCAACAACTCTCAATACCATTTCCACCTGTTTAATCAATTACAAAGTCAGTAACTTCTCTGTTTACCCAACAAGGAATCATGCTGGTAATAGTTACTATAACTTGCTTGATTTCTCCATTCAGAATCTCCGATTCGCAGCGTGCTCTAAACCAAAACCAACTGTCATTATCGTACCAGAGAGCAAGGAGCAGCTGGTGAGCAGCGTTCTGTGTTGCAGACAAGGCTCGTATGAAATCAGAGTAAGGTGCGGTGGACACAGTTATGAAGGGACTTCATCAGTTTCCTTTGATGGTTCCCCATTTGTGGTCATTGATTTGATGAAATTAGACGGCGTTTCAGTGGATGTGGATTCAGAAACCGCGTGGGTACAGGGCGGCGCTACACTTGGCCAGACTTATTATGCCATTTCCCGAGCCAGCAACGTTCATGGATTTTCAGCTGGTTCTTGCCCAACAGTTGGGGTTGGCGGGCACATTTCCGGGGGTGGTTACGGATTTTTATCCAGAAAATATGGACTTGCTGCTGATAACGTGGTGGATGCTCTTCTTGTTGATGCGGAAGGACGGCTATTAGACCGCAAAGCCATGGGAGAAGAAATCTTTTGGGCCATCAGAGGTGGAGGTGGAGGAATTTGGGGAATCATTTACGCCTGGAAAATCCGATTGCTCAAAGTGCCCAAGACCGTGACCAGTTTCATAATCCCTAGGCCTGGCTCCAAACGATATGTGTCCCAACTAGTTCACAAATGGCAACTTGTTGCACCAAAGTTAGAGGATGAATTTTATCTATCGATCTCCATGAGCTCTCCTAGTAAAGGAAACATTCCTATTGAAATAAATGCCCAATTCAGCGGATTTTACCTAGGTACAAAAACCGAAGCCATTTCCATCTTGAATGAGGCCTTTTCGGAGTTGGGAGTTCTGGAAGGTGACTGCAAAGAAATGAGTTGGATTGAATCAACACTTTTCTTCTCCGAATTAAATGACGTTGCGAATTCCTCCGATGTCTCTCGTTTGAAAGAGCGTTACTTTGAAAACAAATCATACTTCAAAGCCAAATCAGACTATGTGAAGACCCCAATTTCAGTGGGTGGGATTATGACGGCTCTTAATGTTCTTGAGAAAGAACCCAACGGACATGTCATCTTGGACCCTTATGGTGGAGCCATGCAAAGAATTAGTGAGGAAGCTATTGCTTTCCCTCATAGAAAGGGTAACCTTTTCGGAATTCAATATCTAGTAGTGTGGAAAGAAAAGGACAATAATAATATTGTCAAGAGCAATATTGGGTACATAGAGTGGATAAGAGAGTTTTACAATACAATGGCACCCCATGTTTCAAGTTCACCTAGGGCAGCTTATGTCAACTACATGGATCTGGACCTTGGAGTGATGGACGACTACTTATTGCCATGTACTAGTACTACTGCGTCTGCTAATCATGCCGTGGAGAGAGCAAGGGTCTGGGGTGAAAAGTATTTCTTGAATAACTATGATAGATTGGTCAAAGCTAAGACAAAAATTGACCCACTAAACGTTTTTCGACATCAACAGGGCATCCCTCCTTTGTTCGCCTCAATGCAAGAGTATACCTATAGTAGTAAA TG APredicted amino acid sequence SEQ ID NO: 4MKRNISMFLQLLLIILMMISFLFTSLLVPSVSATTLNTISTCLINYKVSNFSVYPTRNHAGNSYYNLLDFSIQNLRFAACSKPKPTVIIVPESKEQLVSSVLCCRQGSYEIRVRCGGHSYEGTSSVSFDGSPFVVIDLMKLDGVSVDVDSETAWVQGGATLGQTYYAISRASNVHGFSAGSCPTVGVGGHISGGGYGFLSRKYGLAADNVVDALLVDAEGRLLDRKAMGEEIFWAIRGGGGGIWGIIYAWKIRLLKVPKTVTSFIIPRPGSKRYVSQLVHKWQLVAPKLEDEFYLSISMSSPSKGNIPIEINAQFSGFYLGTKTEAISILNEAFSELGVLEGDCKEMSWIESTLFFSELDDVANSSDVSRLKERYFENKSYFKAKSDYVKTPISVGGIMTALNVLEKEPNGHVILDPYGGAMQRISEEAIAFPHRKGNLFGIQYLVVWKEKDNNNIVKSNIGYIEWIREFYNTMAPHVSSSPRAAYVNYMDLDLGVMDDYLLPCTSTTASANHAVERARVWGEKYFLNNYDRLVKAKTKIDPLNVFRHQQGIPPLFASMQEYTYSSK  BBLd-2 SEQ ID NO: 2ATG AAACGAAATATATCCATGTCTCTTCAGCGTTTGCTCATAATTCTGATGATGATCAGCTTCTTATTTACTTCTCTTCTTGTACCTTCCGTCTCTGCTACAAATCTCAATACCATTTCCACCTGTTTGATCAATTACAAAGTCAGTAACTTCTCTGTTTATCCAACAAGGAATCATGCTGGTAATAGGTACTATAACTTGCTTGATTTCTCCATTCAGAATCTCCGATTCGCAGCGTCCTCTAAACCAAAACCAACGGTCATTATCGTACCAGAGAGCAAGGAGCAGCTGGTGAGCAGCGTTCTGTGTTGCAGACAAGGTTCTTATGAAATCAGAGTAAGGTGCGGAGGACACAGTTATGAAGGGACTTCTTACGTTTCCTTTGATGGTTCCCCATTTGTGGTCATTGATTTGATGAAATTAGATGATGTTTCGGTAGATTTGGATTCCGAAACCGCGTGGGTACAAGGTGGCGCTACACTTGGCCAGACTTATTATGCCATTTCCCGGGCCAGTGACGTTCATGGATTTTCAGCTGGTTCTTGCCCAACAGTTGGGGTTGGGGGCCACATTTCCGGGGGTGGCTTTGGATTTTTATCAAGAAAATATGGACTTGCTGCTGATAACGTGGTGGATGCTCTTCTTGTTGATGCGGAAGGACGGCTATTAGACCGCAAAGCCATGGGAGAAGAAGTGTTTTGGGCCATCAGAGGTGGTGGTGGAGGAATTTGGGGAATCATTTACGCCTGGAAAATCCGATTGCTCAAAGTGCCCAAGACTGTGACTAGTTTCATAGTCCCTAGGCCTGGCTCCAAACGATATGTGTCCCAACTAGTTCACAAATGGCAACTTGTTGCACCAAAGTTAGACGATGACTTTTATCTATCGATCTCCATGAGCTCTGCTAGTAAAGGAAACATTCCTATTGAAATAAATGCCCAATTCAGCGGATTTTACCTAGGTACAAAAACCGAAGCCATTTCCATCTTGAATGAGGCCTTTCCGGAGTTGGGAGTTGTGGAAAGTGACTGCAAAGAAATGAGTTGGATTGAATCAACACTTTTCTTCTCCGAATTAGATAACGTTGCGAACACCTCCGATGTCTCTCGTTTGAAAGAGCGTTACTTTGAAAACAAATCATACTTCAAAGCCAAATCAGACCATGTGAAGACCCCAATTTCAGTGGGAGGGATTATGACAGCTCTTGATGTTCTTGAGAAAGAACCAAATGGACATGTCATCTTTGACCCTTATGGTGCAGCCATGCAGAGAATTAGCGAGGAAGCTATTGCTTTCCCTCATAGAAAGGGTAACCTATTCAGAATTCAATATCTAGTAGTGTGGAAAGAAAAGGACAATAATAATATTGCCAAGAGCAATGGGTACATAGAGTGGATAAGAGAGTTTTACAATACAATGGCACCCCATGTTTCTAGTTCACCTAGGGCAGCTTATGTCAACTATATGGATCTGGACCTTGGAGTGATGGACGACTACTTAATGCTAAATACTAGTATTACTGCCTCTGCTGATCATGCCGTGGAGAGAGCAAGGGTCTGGGGTGAAAAGTATTTCTTGAATAACTATGATAGATTGGTCAAAGCTAAGACAAAAATTGACCCACTAAACGTTTTTCGACATCAACAGGGCATCCCTCCTATGTTCGCCTCAATGCCAGAGCATACCTATAGTAGTAAA TGA  Predicted amino acid sequence SEQ ID NO: 5MKRNISMSLQRLLIILMMISFLFTSLLVPSVSATNLNTISTCLINYKVSNFSVYPTRNHAGNRYYNLLDFSIQNLRFAASSKPKPTVIIVPESKEQLVSSVLCCRQGSYEIRVRCGGHSYEGTSYVSFDGSPFVVIDLMKLDDVSVDLDSETAWVQGGATLGQTYYAISRASDVHGFSAGSCPTVGVGGHISGGGFGFLSRKYGLAADNVVDALLVDAEGRLLDRKAMGEEVFWAIRGGGGGIWGIIYAWKIRLLKVPKTVTSFIVPRPGSKRYVSQLVHKWQLVAPKLDDDFYLSISMSSASKGNIPIEINAQFSGFYLGTKTEAISILNEAFPELGVVESDCKEMSWIESTLFFSELDNVANTSDVSRLKERYFENKSYFKAKSDHVKTPISVGGIMTALDVLEKEPNGHVIFDPYGAAMQRISEEAIAFPHRKGNLFRIQYLVVWKEKDNNNIAKSNGYIEWIREFYNTMAPHVSSSPRAAYVNYMDLDLGVMDDYLMLNTSITASADHAVERARVWGEKYFLNNYDRLVKAKTKIDPLNVFRHQQGIPPMFASMPEHTYSSK  BBLe SEQ ID NO: 3 ATGTTTCCAATCATAATTCTGATCAGCTTTTCATTTACTTTCCTCTTTGCTAGTGTTACTAGTGGAGCAGGAGGAGTTACAAATCTTTCCACCTGTTTAATCAACCACAATGTCCATAACTTCTCTATTTACCCCACAAAGAATGATCAAAGTAGTAGTAATTACTTTAACTTGCTCGATTTTTCCCTTCAGAATCTTCGATTTGCTGCATCTTACATGCCGAAACCAACGGTCATTATCCTACCAAACAGCAAAGAGGAGCTCGTGAGTACCATTCTTTGTTGCAGACAAACATCTTATGAAATCAGAGTAAGGTGCGGAGGACACAGTTACGAGGGAACTTCTTACGTTTCCTTTGACGGTTCCCCTTTCGTGATCGTTGACTTGATGAAATTAGACGACGTTTCAGTAGATTTGGATTCCGAAACAGCTTGGGCTCAGGGCGGCGCAACAATTGGCCAAATTTATTACGCCATTTCCAGGGTTAGTGACGTTCATGCATTTTCAGCAGGTTCGGGACCAACAGTAGGATCTGGAGGTCATATTTCAGGTGGCGGCTTTGGACTAATGTCCAGAAAATTCGGACTCGCTGCTGATAGTGTCGTTGATGCTCTTCTAATTGATGCTGAAGGACGGTTATTAGACCGGAAAGCCATGGGAGAAGACGTATTTTGGGCAATCAGAGGTGGCGGCGGTGGAAATTGGGGAATTATTTATGCCTGGAAAATTCGATTACTCAAAGTGCCTAAAATCGTAACAACTTGTATGATCTATAGGCCTGGATCCAAACAATACGTGGCTCAACTACTTCAGAAATGGCAAATAGTTACTCCAAATTTGGCCGATGATTTTACTCTAGGAGTACTCATGAGACCTATAGATCTGCGGGCGGATATGAATTACGGAAATACTACTCCTATTGAAACATTTCCCCAATTCAATGCACTTTATTTGGGTCCAAAAACTGAAGCGGTTTCCATATTAAATGAGGCATTTCCAGAGCTGGACGCTAAGAATGATGACGCCAAAGAAATGACTTGGATTGAGTCAGCACTTTTCTTTTCCGAATTAGATAACGTATTCGGGAACTCCTCTGACGATATCTCCCGTTTGAAAGAACGCTACATGGACGCAAAAACTTTCTTCAAAGGCAAATCAGATTTTGTGAAGACTCCATTTTCAATGGACGCGATGATGACAGCTCTTGTTGAACTCGAGAAAAACCCCAAGTCATTCCTTGTCTTCGATCCTTATGGCGGAGTCATGGACAAGATTAGTGATCAAGCTATTGCTTTCCCTCATCGAAAGGGTAACCTTTTCGCGGTTCAATATTATGCATTTTGGAACGAAGAGGACGATGCCAAGAGCAACGAGTACATAGAGTGGACAAGGGGATTTTACAATAAAATGGCGCCTTTTGTTTCAAGCTCGCCAAGGGGAGCTTATATCAACTACTTGGATATGGATCTTGGAGTGAATATGGACGACGACTACTTACTGCGAAATGCTAGTAGTCGTAGTTCTTCTTCCTCTGTTGATGCTGTGGAGAGAGCTAGAGCGTGGGGTGAAATGTATTTCTTGCATAACTATGATAGGTTGGTTAAAGCTAAGACACAAATTGATCCACTAAATGTTTTTCGACATGAACAGAGTATACCTCCTATGCTTGGTTCAACGCAAGAGCACAGTAGTGAA TGA  Predicted amino acid sequence SEQ ID NO: 6MFPIIILISFSFTFLFASVTSGAGGVTNLSTCLINHNVHNFSIYPTKNDQSSSNYFNLLDFSLQNLRFAASYMPKPTVIILPNSKEELVSTILCCRQTSYEIRVRCGGHSYEGTSYVSFDGSPFVIVDLMKLDDVSVDLDSETAWAQGGATIGQIYYAISRVSDVHAFSAGSGPTVGSGGHISGGGFGLMSRKFGLAADSVVDALLIDAEGRLLDRKAMGEDVFWAIRGGGGGNWGIIYAWKIRLLKVPKIVTTCMIYRPGSKQYVAQLLQKWQIVTPNLADDFTLGVLMRPIDLRADMNYGNTTPIETFPQFNALYLGPKTEAVSILNEAFPELDAKNDDAKEMTWIESALFFSELDNVFGNSSDDISRLKERYMDAKTFFKGKSDFVKTPFSMDAMMTALVELEKNPKSFLVFDPYGGVMDKISDQAIAFPHRKGNLFAVQYYAFWNEEDDAKSNEYIEWTRGFYNKMAPFVSSSPRGAYINYLDMDLGVNMDDDYLLRNASSRSSSSSVDAVERARAWGEMYFLHNYDRLVKAKTQIDPLNVFRHEQSIPPMLGSTQEHSSE 

In silico analyses of EST databases can serve as a useful indicator topredict the relative contribution of individual members of a gene familytoward the overall transcript pool. The results of our in silicoanalysis of the BBL family is shown below in Table 3.

TABLE 3 In silico analysis of BBL gene expression. BBL EST LibraryRoot-Specific Isoform Hits ESTs^(a) BBLa 38 38 BBLb 11 10 BBLc 16 16BBLd-1 2 1 BBLe 4 4 ^(a)In addition to cDNA from libraries generatedsolely from root tissue, cDNAs isolated from pooled leaf, flower, androot cDNA libraries were also included.

Consistent with the characterization of the BBL gene family reported byKajikawa et al. (2011), the in silico analysis suggests that BBLa, BBLband BBLc are the most highly transcribed members of this gene family.This is also consistent with the dramatic reduction in alkaloid contentobserved in tobacco plants mutated at these three loci (Table 2).Although fewer ESTs were found corresponding to BBLd-1 and BBLe, theirpresence in the EST database proves that they are in fact transcribedand are therefore likely to be contributing toward the overall BBLactivity of the tobacco plant. This is particularly true for BBLe givenits greater sequence similarity to BBLa, BBLb and BBLc, as well as itsincreased representation in the in silico analysis compared to BBLd-1.Although no ESTs were found that corresponded to BBLd-2, we cannot ruleout the possibility that this isoform is also transcribed at a low leveland thereby contributes toward overall cellular BBL activity.

The work described in Example 1 and that reported in Lewis et al. (2015)demonstrate that it is possible to produce low alkaloid tobacco plantsthat maintain favorable quality properties by inhibiting the function ofgenes of the BBL family. Despite the substantial progress that has beenmade in this area, the existing technology still falls short of whatwould be optimal for many low nicotine tobacco applications. The primarydeficiency is that the alkaloid levels remaining in the current art arestill not as low as would be desired for certain applications. Forexample, the cured leaf of the RNAi line displaying the greatestreduction in nicotine content in Table 1 (DH32) still possessed about17% of the nicotine observed in the normal K326 plants. For theEMS-mutagenized DH98 325-6 materials, the triple mutant bbl plantsretained about 7.5% of the total alkaloid content observed in the nullsegregant controls. There remains a need to develop tobacco cultivarsthat produce and accumulate even lesser quantities of nicotine withinhigh quality tobacco backgrounds than has been previously described, oris even possible using the existing art.

Example 3. Targeted Mutagenesis of BBL Genes

Another deficiency in the current technology has been the reliance onnon-target-specific mutagens such as EMS in order to generate genemutations in plants for situations where it is preferable that the endproduct be non-transgenic. Mutagens like EMS function to randomlydistribute mutations throughout the entire genome. In order to obtain areasonable likelihood of finding a mutation in a gene of interest usingnontarget-specific agents, one must treat the plant in a manner wherebymutations are incorporated at a high density throughout the entiregenome. Once a mutation in a gene of interest is identified in a plantfrom such a mutagenized population, extensive backcrossing is thenrequired to attempt to eliminate all the undesirable mutationsdistributed throughout the genome that will be found in that plant aswell. Should an undesirable secondary mutation be closely linked to themutation of interest, it can become difficult to separate it from thedesired mutant trait. A problem frequently encountered using randommutation breeding approaches is that the linked deleterious secondarymutations prevents incorporation of the mutant trait of interest intohigh quality commercial cultivars in a manner that does not negativelyaffect important agronomic and/or quality traits. With the recent adventof molecular biology-based precision mutagenesis technologies, it is nowpossible to induce mutations into a specific gene or genes of interestwithout introducing unwanted random mutations throughout the genome.These technologies promise to greatly increase the ability to introducespecific desired mutations within the genome of a crop of interest in amanner than do not otherwise negatively impact favorable attributes ofthe recipient parent, as well as reduce or eliminate the time involvedwith conducting extensive backcrossing, as is currently required fortraditional mutation breeding approaches.

To date there have been four distinct classes of designer nucleases thathave been developed for the purpose of targeted gene modification ineukaryotes: (1) zinc finger nucleases (ZFNs); (2) custom-designed homingenzymes, or “meganucleases”; (3) transcription activator-like effectornucleases (TALENs); and (4) clustered, regularly interspaced, shortpalindromic repeats (CRISPR)-associated (Cas) nucleases (Puchta andFauser, 2014). When introduced into a plant cell, a custom-designednuclease will bind to its target site(s) and create a double-strandedbreak in the DNA at that location (Curtin et al., 2012; Puchta andFauser, 2014). Plants have two distinct cellular repair mechanisms thatfunction in response to DNA breaks: non-homologous end joining (NHEJ)and homologous recombination (HR). The NHEJ repair system functions byreligating the broken ends, whereas the HR-mediated system utilizes ahomologous sequence as a donor template to repair the break viarecombination. In most plant cells, the NHEJ system predominates.Because NHEJ is error-prone, however, the repeated cutting of a targetsite by a nuclease and religation by NHEJ will lead to a high frequencyof short deletions and/or short insertions at the cut site. When anuclease targets an exon region of a gene, these in-dels frequentlyresult in frame-shift mutations that effectively cause a loss of genefunction. Thus the introduction of a custom-designed nuclease into aplant cell can serve as a powerful tool for knocking out the function ofa targeted gene. Because the introduced transgene carrying the designernuclease will nearly always integrate at a site that is unlinked to thegene(s) being targeted, it can be segregated away in subsequentgenerations of self-fertilization and/or crossing, resulting in plantsin which the targeted endogenous gene(s) have been inactivated, but nolonger carry any foreign DNA.

1. Development of Ultra-Low Nicotine Tobacco Cultivars by TargetedMutagenesis of bbl Family Members Using Custom-Designed Nucleases

A. Constructs Encoding Meganucleases Targeting BBLe, BBLd-1 and BBLd-2

Plasmids encoding meganucleases designed to cleave specific targets inthe BBLe, BBLd-1 and BBLd-2 genes were produced by the company PrecisionBiosciences (Table 4). Construct BBL 1-2x.81 encodes a nuclease that wasengineered to recognize a unique 22 bp sequence within BBLe. Due to thehigh sequence identity shared between BBLd-1 and BBLd-2, it was possibleto design a single nuclease (BBL 7-8x.90) capable of targeting both ofthese genes. For each custom-designed nuclease, a 22 bp target site waschosen that failed to be present anywhere within the recently publishedNicotiana tabacum reference genomes except for the intended gene targets(Sierro et al., 2014). The selection of unique target sites should helpminimize the possibility of off-target cleavage elsewhere in the genome.Furthermore, the selected target sites occur in the upper one third ofthe gene sequences (see sequences in Example 2 above), upstream of thesequences encoding the highly conserved FAD binding domain. Thereforemutation events leading to a frame shift at these locations would bepredicted to produce completely nonfunctional protein products.Constructs BBL 1-2x.81 and BBL 7-8x.90 were cloned into the plantexpression vector pCAMBIA2300 (www.cambia.org). Within pCAMBIA2300,transcription of the designer nucleases is driven by an enhanced 35SCauliflower Mosaic Virus promoter, and selection is mediated via thenptII gene that confers resistance to the antibiotic kanamycin.

TABLE 4 Custom-designed nuclease constructs that  target BBL genes.Target  Designer Gene Target Site Nuclease BBLe GAGGAGCTCGTGAGTACCATTCBBL  (SEQ ID NO: 7) 1-2x.81 BBLd-1,  GTCATTATCGTACCAGAGAGCA BBL  BBLd-2(SEQ ID NO: 8) 7-8x.90

B. Targeted Mutagenesis of BBLe, BBLd-1 and BBLd-2 within a TripleMutant bbla, bblb and bblc Background

To determine whether introducing mutations in the BBLe, BBLd-1 and/orBBLd-2 genes can reduce nicotine levels below that which is attainablein plants homozygous for knockout mutations in the three previouslycharacterized BBL genes (BBLa, BBLb and BBLc), constructs BBL 1-2x.81and BBL 7-8x.90 were introduced into line TN90 (bbla/bblb/bblc) usingstandard Agrobacterium-mediated transformation protocols (Horsch et al.,1985). TN90 (bbla/bblb/bblc) was produced by backcrossing an originaltriple mutant bbla/bblb/bblc individual in its EMS-mutagenized DH98325-6 background (Lewis et al., 2015) into the commercial burleycultivar TN90. Seven generations of backcrossing were conducted to bringthe TN90 recurrent parent back to type and to vastly reduce the numberof genome-wide EMS mutations present in the starting DH98 325-6(bbla/bblb/bblc) materials. With each backcross generation, SNP markersspecific for the debilitating bbla, bblb and bblc mutations were used toidentify progeny carrying the desired bbl loci; upon completion of sevenbackcross generations, BC₇F₂ individuals were screened with the samemarkers to identify line TN90 (bbla/bblb/bblc) that is homozygous forall three bbl mutations.

Over 150 kanamycin resistant T₀ plants were screened for mutations inBBLe, BBLd-1 or BBLd-2. Mutation screening was conducted by isolatinggenomic DNA from the leaves of very young T₀ plants and amplifying theregions of interest through PCR using primers flanking the targeted cutsites. The resulting PCR products were subsequently analyzed by DNAsequence analysis. If a genome editing event occurred soon aftertransformation and prior to the first cell division, this can be readilydetected on a sequence chromatogram as these sequences will diverge fromwild type (WT) at the edited site. If there are two distinct sequencingpatterns of equal intensity at the site of sequence divergence, one WTand the other edited, we classify this plant as being “monoallelic”, asonly one of the two alleles of the target gene would appear to have beenmutated. Plants possessing a single edited sequence pattern, or twooverlapping patterns that both differ from WT are designated “biallelic”events. Given the large number of cells that comprise a young tobaccoleaf, editing events that occurred at a late-stage post-transformationand are only present in a small number of cells would not be detectedusing this screening method. Plants containing late event mutationswould be considered “chimeric” and are of lesser interest as theseediting events would be much less likely to be present in the germlineand inherited by the progeny.

T₀ plants containing genome editing events mediated by constructsBBL1-2x.81 or BBL7-8x.90 are shown in FIG. 1 . Nuclease-mediated editingis manifest as short deletions and/or insertions emanating from thepredicted cut site of the enzyme. Of the eight T₀ plants identified withmutations in BBLe, seven were monoallelic for the mutation and one wasbiallelic (plant #143). Eight T₀ plants were also identified thatdisplayed nuclease-induced mutations in BBLd-1, all of which werepredicted to be monoallelic. For BBLd-2, nine monoallelic events and onebiallelic event (plant #36) were observed amongst the T₀ plantsscreened. Two plants transformed with the BBL7-8x.90 construct possessedmutations in both BBLd-1 and BBLd-2 (#36 and #100). Overall,approximately 18% of the T₀ plants transformed with BBL1-2x.81 orBBL7-8x.90 screened positive for mutations at the intended BBL loci.This level of mutation efficiency is similar to that reported by otherswho have used custom-designed meganuclease enzymes for targetedmutagenesis in plants (Gao et al., 2010; Dewey and Lewis, 2014). Ofparticular interest are plants harboring deletion/insertion mutations ina BBLe, BBLd-1 and BBLd-2 gene whose net loss or gain of nucleotides isa number that is not divisible by three. Mutations of this nature wouldbe predicted to lead to a completely nonfunctional protein product, asthe reading frame of the gene sequences upstream of the mutation sitewould not be in frame with those downstream of the mutation. T₀ eventsthat fit this description are underlined in FIG. 1 .

2. Evaluation of Plants with BBLe, BBLd-1 and BBLd-2 Mutations

To transgenics containing targeted mutations of interest are crossedand/or self-fertilized to produce progeny homozygous for the variouspossible mutant combinations and that have also segregated away themutagenic transgene(s). Plants that lack any foreign DNA and carryvarious combinations of bbld-1, bbld-2 and bble mutations (all stillwithin a triple mutant bbla/bblb/bblc background) are grown and curedaccording to standard industry practice and assayed for alkaloidcontent. Comparisons are made to the relevant original wild typegenotype (e.g. TN90), as well as its relevant parental genotypehomozygous for only the bbla, bblb and bblc mutant loci (e.g. TN90(bbla/bblb/bblc)). In addition, all plants are evaluated for grade indexand percent reducing sugars. The outcome is the development of highquality tobacco cultivars in commercially viable backgrounds thataccumulate significantly less nicotine than their respective parentalplants (bblabbla/bblbbblb/bblcbblc) due to the introduction andpyramiding of knockout mutations in the BBLe and/or BBLd-1 and/or BBLd-2loci.

The foregoing is illustrative of the invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

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1.-28. (canceled)
 29. A method of producing a tobacco product comprisingtobacco from a Nicotiana plant, the method comprising: (1) combining ina Nicotiana plant: (A) a modification that reduces the activity of atleast one of BBLa, BBLb, and BBLc or reduces the expression of a nucleicacid encoding at least one of BBLa, BBLb, and BBLc; and (B) a mutationin at least one endogenous polynucleotide or combination ofpolynucleotides selected from the group consisting of: (i) apolynucleotide having about 97% to about 100% sequence identity to SEQID NO:1 and a polynucleotide having about 97% to about 100% sequenceidentity to SEQ ID NO: 2; (ii) a polynucleotide having about 97% toabout 100% sequence identity to SEQ ID NO: 1 and a polynucleotide havingabout 97% to about 100% sequence identity to SEQ ID NO: 3; (iii) apolynucleotide having about 97% to about 100% sequence identity to SEQID NO:2; and (iv) a polynucleotide having about 97% to about 100%sequence identity to SEQ ID NO:3, such that the Nicotiana plant has anicotinic alkaloid content that is reduced as compared to a plant thatis not modified per (A) and does not comprise the mutations of (B); and(2) producing a tobacco product from the Nicotiana plant.
 30. The methodof claim 29, wherein the Nicotiana plant is modified so as to reduce theactivity of at least two of BBLa, BBLb, and BBLc or to reduce expressionof a nucleic acid encoding at least two of BBLa, BBLb, and BBLc.
 31. Themethod of claim 30, wherein the Nicotiana plant is modified so as toreduce the activity of each of BBLa, BBLb, and BBLc or to reduceexpression of a nucleic acid encoding each of BBLa, BBLb, and BBLc. 32.The method of claim 31, wherein the Nicotiana plant comprises a mutationin each of at least two of the polynucleotides or the polynucleotidecombinations of (i)-(iv).
 33. The method of claim 32, wherein theNicotiana plant comprises a mutation in each of the polynucleotides orthe polynucleotide combinations of (i)-(iv).
 34. The method of claim 31,wherein the Nicotiana plant has a nicotinic alkaloid content that isreduced by at least about 90% as compared to a plant that is notmodified per (A) and does not comprise the mutations of (B).
 35. Themethod of claim 34, wherein the Nicotiana plant has a nicotinic alkaloidcontent that is reduced by at least about 95% as compared to a plantthat is not modified per (A) and does not comprise the mutations of (B).36. The method of claim 34, wherein the Nicotiana plant has a nicotinicalkaloid content that is reduced by at least about 97% as compared to aplant that is not modified per (A) and does not comprise the mutationsof (B).
 37. The method of claim 29, wherein the Nicotiana plant: (a) ismodified so as to reduce the activity of each of BBLa, BBLb, and BBLc orto reduce expression of a nucleic acid encoding each of BBLa, BBLb, andBBLc, and (b) comprises a mutation in at least polynucleotide (iv), andwherein the nicotinic alkaloid content of the Nicotiana plant is reducedby at least about 90% as compared to a plant that is not modified per(A) and does not comprise the mutations of (B).
 38. The method of claim29, wherein the tobacco product is selected from the group consisting ofleaf tobacco, shredded tobacco, cut tobacco, ground tobacco, powdertobacco, tobacco extract, smokeless tobacco, moist or dry snuff, pipetobacco, cigar tobacco, cigarillo tobacco, cigarette tobacco, andchewing tobacco.
 39. The method of claim 29, wherein the tobacco productis selected from the group consisting of a cigarillo, a kretekcigarette, a non-ventilated recess filter cigarette, a vented recessfilter cigarette, an electronic cigarette, heat not burn products, acigar, snuff, tobacco-containing gum, tobacco-containing lozenges, andchewing tobacco.
 40. The method of claim 29, wherein the nicotinicalkaloid is nicotine.
 41. The method of claim 29, wherein producing thetobacco product comprises curing the Nicotiana plant.
 42. The method ofclaim 41, wherein the curing step is performed by air curing, groundcuring, rack curing, pit curing, fire curing, sun curing, flue curing,or any combination thereof.